Impact of Endothelium-Derived Mitochondrial Reactive Oxygen Species on Obesity-Associated Atherosclerosis

Abstract

Aims:

Obesity, a risk factor for atherosclerosis development and progression, is marked by excessive reactive oxygen species (ROS) production. We previously demonstrated that high-glucose (HG) conditions induce mitochondrial ROS (mtROS) production in aortic endothelial cells (ECs). However, the link between elevated mtROS levels in obesity and atherosclerosis progression remains unclear. This study aimed to investigate whether endothelial-specific mtROS suppression by overexpressing manganese superoxide dismutase (MnSOD) could attenuate atherosclerosis progression in high-fat diet (HFD)-induced obese apolipoprotein E-deficient (ApoE KO) mice.

Results:

Atherosclerotic lesion formation did not differ significantly between normal chow-fed control ApoE KO mice and endothelial cell-specific MnSOD-overexpressing ApoE KO (eMnSOD-Tg/ApoE KO) mice. However, in HFD-fed groups, eMnSOD-Tg/ApoE KO mice exhibited reduced atherosclerotic lesion size, decreased relative ROS levels, and lower Icam1 and Ccl2 expression compared to HFD-fed control ApoE KO mice. In obese ApoE KO mice, serum glucose, lipopolysaccharide (LPS), and low-density lipoprotein (LDL) levels were elevated. In human aortic endothelial cells, exposure to LPS, oxidized LDL, and high glucose increased relative mtROS levels, which was effectively attenuated by MnSOD overexpression. MnSOD also suppressed intercellular adhesion molecule-1 and monocyte chemoattractant protein-1 expression under these conditions.

Innovation:

Our findings demonstrate that endothelial-specific MnSOD overexpression suppresses obesity-related atherosclerosis in ApoE KO mice.

Conclusion:

mtROS plays a pivotal role in obesity-associated atherosclerosis, and targeting endothelial mtROS may offer a therapeutic strategy for preventing vascular complications in obesity. Antioxid. Redox Signal. 45, 63–77.

Graphical Abstract

Keywords

atherosclerosis endothelium manganese superoxide dismutase mitochondrial reactive oxygen species obesity

Introduction

The prevalence of obesity is increasing worldwide (NCD Risk Factor Collaboration, 2019; Finucane et al., 2011). Obesity increases the risk of diseases such as type 2 diabetes, fatty liver disease, hypertension, myocardial infarction, stroke, and obstructive sleep apnea syndrome, thereby reducing the quality of life of individuals and causing a medical and economic burden on society. Therefore, elucidating the pathogenesis of obesity-related diseases, particularly atherosclerotic diseases, such as myocardial infarction and stroke, will contribute to reducing obesity-related mortality and maintaining quality of life.

Obesity is the result of a complex interaction between genetic and environmental factors. Among environmental factors, an increase in fat intake due to changes in diet increases metabolic abnormalities, such as insulin resistance and elevated serum lipid levels, which are characteristic of metabolic syndrome (MetS). Furthermore, MetS is characterized by excessive reactive oxygen species (ROS) production (Roberts and Sindhu, 2009). The interplay between obesity and oxidative stress is multifaceted. Oxidative stress contributes to chronic inflammation pathogenesis, increased ROS production, mitochondrial dysfunction, antioxidant defense system dysregulation, and various metabolic abnormalities. Obesity promotes oxidative stress, which in turn plays a critical role in the development of metabolic disorders and obesity-associated diseases (Balan et al., 2024). However, the details of the involvement of mitochondrial ROS (mtROS) in the pathology of obesity and MetS, particularly in atherosclerotic disease development, are unclear.

The mechanisms of ROS production in the body include the mitochondrial respiratory chain, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, xanthine oxidase, lipoxygenase, non-coupled nitric oxide synthase (NOS), and myeloperoxidase oxidase. Research into cardiovascular disease focusing on these has developed rapidly in recent years (Sugamura and Keaney, 2011). ROS contributes to cardiovascular disease (CVD) development by damaging DNA and proteins by promoting inflammation in tissues (Austin et al., 2016). Furthermore, increasing evidence suggests that chronic and acute ROS overproduction under pathophysiological conditions is essential for CVD development (Madamanchi et al., 2005). An imbalance between ROS production and detoxification leads to increased oxidative stress within the cell. Superoxide dismutase (SOD) scavenger enzymes are the main ROS detoxifying enzymes (Fridovich, 1995). Of these SODs, manganese SOD (MnSOD) is an essential mitochondrial antioxidant enzyme that detoxifies free radical superoxide, a necessary byproduct of mitochondrial respiration (Nishikawa et al., 2000; Sarsour et al., 2014).

Innovation

This study is the first to demonstrate that obesity induced by an HFD may accelerate atherosclerosis progression via enhanced mtROS production, triggered by Ox-LDL, LPS, and hyperglycemia. Furthermore, our findings suggest that mtROS generation in vascular endothelial cells (induced by specific pathological stimuli) promotes the expression of adhesion molecules and other atherogenic factors, thereby contributing to vascular inflammation and plaque development. These results highlight a potential mechanistic pathway by which obesity exacerbates atherosclerosis and suggest that targeting endothelial mtROS may represent a novel therapeutic strategy for preventing atherosclerotic diseases.

Studies on mice with heterozygous MnSOD deficiency have shown that atherosclerosis is accelerated in these mice (Ohashi et al., 2006; Vendrov et al., 2017), and MnSOD may act to suppress atherosclerosis progression. Furthermore, in a study of patients with type 2 diabetes, 8-hydroxydeoxyguanosine (8-OHdG), which is used as an indicator of intracellular oxidative stress, including mtROS, increased in patients with advanced atherosclerosis. Additionally, 8-OHdG is low in patients who maintain reasonable blood glucose control over a long period, correlating with the thickening of the carotid intima-media complex (Nishikawa et al., 2003). In contrast, several reports suggest that oxidative stress and mitochondrial dysfunction may contribute to the progression of atherosclerosis through the reduction of vascular endothelial function and adhesion molecule expression (Ballinger, 2005; Dorighello et al., 2016; Hulsmans et al., 2012; Madamanchi et al., 2005). However, how regulating mtROS specifically in vascular endothelial cells affects the progression of atherosclerosis remains unclear.

We previously reported that hyperglycemia induces mtROS production and subsequently induces the activation of the polyol pathway, formation of advanced glycation end products, and activation of protein kinase C, which are considered to be factors in diabetic vascular complications (Nishikawa et al., 2000). Furthermore, we reported that mtROS is associated with the development of diabetic microvascular complications, such as diabetic nephropathy and retinopathy, and that MnSOD-based mtROS inhibition inhibits their progression (Goto et al., 2008; Sada et al., 2016; Yoshinaga et al., 2021). However, the relationship between mtROS regulation and atherosclerosis development and progression remains unknown.

Apolipoprotein-E knockout (ApoE KO) mice are a well-established model of atherosclerosis, as they spontaneously develop hypercholesterolemia and atherosclerotic lesions due to impaired clearance of remnant lipoproteins. This model has therefore been widely used to investigate the molecular mechanisms underlying atherosclerosis (Getz and Reardon, 2012; Zhang et al., 1992). Based on this background, this study aimed to determine whether endothelial-specific regulation of mtROS could inhibit the progression of atherosclerosis in obese mice fed a high-fat diet (HFD). To address this question, we used ApoE KO mice with endothelial cell-specific overexpression of MnSOD, a key mitochondrial superoxide scavenging enzyme. We found that MnSOD overexpression in vascular endothelial cells significantly suppressed atherosclerosis progression in obese ApoE KO mice fed an HFD without affecting body weight, blood glucose levels, or serum lipid profiles. In addition, when we analyzed oxidized low-density lipoprotein (Ox-LDL), lipopolysaccharide (LPS), and hyperglycemia, which are assumed to be factors that induce atherosclerosis when an HFD is loaded, using human aortic endothelial cells (HAECs), we found that all of these factors increased the expression of monocyte chemoattractant protein 1 (MCP-1) and adhesion molecules, as well as relative mtROS levels. Finally, these effects were suppressed by MnSOD overexpression.

Results

To investigate the effect of EC-specific MnSOD overproduction in atherosclerotic lesion formation, we developed EC-specific MnSOD-overproducing and apolipoprotein-E-deficient (eMnSOD-Tg/ApoE KO) mice. These mice (aged 12 weeks) were fed with a normal-chow diet (NCD) or high fat diet (HFD) for 8 weeks. Casual blood glucose levels, body weight, and serum lipid profiles did not differ between control ApoE KO and eMnSOD-Tg/ApoE KO mice (Supplementary Fig. S1). Atherosclerotic lesion formation between NCD-fed control ApoE KO and eMnSOD-Tg/ApoE KO mice did not differ (Fig. 1, A–D). However, atherosclerotic lesion formation was decreased in HFD-fed eMnSOD-Tg/ApoE KO mice compared to that in control HFD-fed ApoE KO mice (Fig. 1, E–H). Immunochemistry assays revealed the presence of F4/80-positive cells in the atherosclerotic lesions of HFD-fed ApoE KO mice (Fig. 2A, upper panels). However, the number of F4/80-positive cells was reduced in the atherosclerotic lesions of HFD-fed eMnSOD-Tg/ApoE KO mice compared to control HFD-fed ApoE KO mice (Fig. 2A, upper panels, and 2B). Moreover, the 8-OHdG-positive area was also reduced in the atherosclerotic lesions of HFD-fed eMnSOD-Tg/ApoE KO mice (Fig. 2A, middle panels, and 2C). Furthermore, the number of F4/80- and 8-OHdG-double positive cells was reduced in the atherosclerotic lesions of HFD-fed eMnSOD-Tg/ApoE KO mice (Fig. 2A, lower panels, and 2D). Hematoxylin and eosin staining revealed that the area of necrotic core, defined as acellular and anuclear regions within the plaque, was significantly reduced in eMnSOD-Tg/ApoE KO mice compared with that in control ApoE KO mice (Fig. 2E and F). In addition, Masson’s trichrome staining showed increased collagen deposition within the plaques of eMnSOD-Tg/ApoE KO mice, indicating enhanced fibrotic content (Fig. 2G and H). In contrast, the mRNA expression levels of Ccl2 (C-C motif chemokine ligand 2; also known as MCP-1) and Icam1 (intercellular adhesion molecule 1; ICAM-1) were significantly decreased in eMnSOD-Tg/ApoE KO mice, whereas Vcam1 (vascular cell adhesion molecule 1; VCAM-1), Sele (selectin, endothelial cell adhesion molecule; also known as E-selectin), and Il6 (interleukin-6; IL-6) expression remained unchanged (Fig. 3).

FIG. 1.

Endothelium-specific MnSOD overexpression suppresses aortic plaque progression in HFD-fed ApoE KO mice. ApoE KO mice or eMnSOD-Tg/ApoE KO mice were treated with normal chow diet (NCD) (A–D) or high-fat diet (HFD) (E–H) for 8 weeks. (A and B: ApoE KO, n = 9 and eMnSOD-Tg/ApoE KO, n = 8, C and D: ApoE KO, n = 7 and eMnSOD-Tg/ApoE KO, n = 7, E and F: ApoE KO, n = 9 and eMnSOD-Tg/ApoE KO, n = 7, and G and H: ApoE KO, n = 15 and eMnSOD-Tg/ApoE KO, n = 11). Representative photomicrographs of Oil Red O-stained fatty lines in the thoracic aorta (A and C) or aortic sinus (E and G) are shown. (B, D, F, and H) Quantitative analysis of atherosclerotic lesion area was presented. Statistical analyses were performed using an unpaired t-test. *: p < 0.05 vs. control mice. Data were presented as mean ± SEM. Scale bars (in yellow), 100 µm. Encircled markings indicate areas of atherosclerotic lesions (A, C, E, and G).

FIG. 2.

Endothelium-specific MnSOD overexpression suppresses oxidative stress and macrophage accumulation within aortic plaques in high-fat diet-fed ApoE KO mice. ApoE KO mice or eMnSOD-Tg/ApoE KO mice were fed a high-fat diet for 8 weeks. (A) Representative fluorescent immunostaining images of the aortic sinus from each group are shown. F4/80 antibody was used to evaluate macrophage infiltration, and 8-hydroxy-2'-deoxyguanosine (8-OHdG) antibody was used to assess oxidative stress levels. Cell nuclei were visualized using DAPI staining. Scale bars (in yellow), 100 µm. (B–D) Data were presented as the number of (B) F4/80-positive cells, (C) 8-OHdG-positive cells, and (D) double-positive cells per plaque area (15 sections, 5 mice per group), and were presented as mean ± SEM. (E) Representative Masson’s trichrome staining images of the aortic sinus in each group are shown. (F) The data presented show the percentage of stained positive area relative to the aortic valve annulus (four animals per group), and were presented as mean ± SEM. Statistical analyses were performed using an unpaired t-test.*p < 0.05. (G) Representative HE staining images of the aortic sinus in each group are shown. (H) The presented data show the percentage of necrotic core area relative to plaque area in the aorta (four animals per group).and were presented as mean ± SEM. Statistical analyses were performed using an unpaired t-test.*p < 0.05.Statistical analyses were performed using an unpaired t-test. *p < 0.05. Encircled markings indicate areas of atherosclerotic lesions (A, C, E, and G).

FIG. 3.

MnSOD overexpression suppresses atherosclerosis-related molecule expression in aortic plaques of high-fat diet-fed mice. ApoE KO mice or eMnSOD-Tg/ApoE KO mice were fed a high-fat diet for 8 weeks. The expression of various molecules in the mRNA of mouse aortae was determined using real-time PCR. Values were normalized to β-actin, and data are presented as the mean ± SEM (ApoE KO, n = 5 and eMnSOD-Tg/ApoE KO, n = 5). Statistical analyses were performed using an unpaired t-test. *p < 0.05.

Next, we investigated the underlying factors of endothelial-specific MnSOD overexpression on atherosclerosis progression under NCD and HFD conditions. Compared to NCD-fed ApoE KO mice, HFD-fed ApoE KO mice exhibited an increased area under the curve for glucose levels in both the intraperitoneal glucose tolerance test and intraperitoneal insulin tolerance test (Supplementary Fig. S2A and S2B). Moreover, serum levels of low-density lipoprotein (LDL) cholesterol (LDL-C), small dense LDL (sdLDL) cholesterol (sdLDL-C), LPS, and insulin were also elevated in HFD-fed ApoE KO mice (Supplementary Fig. S2C–S2F). In addition, the blood pressure (SBP, DBP and MBP) and heart rate (HR) between eMnSOD-Tg mice and control ApoE KO mice after HFD feeding did not differ significantly (Supplementary Fig. S3A–S3D). These findings suggest that hyperglycemia; insulin resistance; increased LDL, sdLDL, LPS serum levels; and insulin contribute to the acceleration of atherosclerosis in HFD-fed ApoE KO mice. Therefore, to identify factors that can increase relative mtROS levels in HAECs, we examined the ability of Ox-LDL, LPS, and insulin (all in addition to glucose), to increase relative mtROS levels. Compared to the normal glucose condition, Ox-LDL and LPS treatment for 3 h, as along with the high glucose condition, increased fluorescence intensity in HAECs, whereas the fluorescence intensity with insulin (1–100 ng/mL) remained unchanged (Fig. 4A and B).

FIG. 4.

Effects of oxidized LDL, LPS, high glucose, and insulin on mitochondrial ROS production. HAECs were stimulated for 3 h with oxidized LDL, LPS, high glucose, or insulin, and mitochondrial ROS production was assessed using 5 µM MitoSOX Red (red) for 20 min. (A) Representative confocal microscopy images (red). (B) Relative fluorescence intensity of MitoSOX Red. Data were presented as the mean ± SEM from four independent experiments conducted in triple form. Statistical analyses were performed using one-way analysis of variance followed by Tukey’s multiple comparison test.*p < 0.05. Ox-LDL: 20 ng/mL, LPS: 10 ng/mL, glucose: 25 mM, Insulin: 1, 10, or 100 ng/mL.

Next, using a MnSOD overexpression HAEC model with adenovirus, we investigated the involvement of mtROS in the atherogenic effects induced by Ox-LDL and LPS. Infection with the MnSOD-overexpressing adenovirus (Ad-MnSOD) increased MnSOD expression at the protein level (Fig. 5A and B). Compared to the lacZ control adenovirus (Ad-lacZ), infection with the Ad-MnSOD reduced both Ox-LDL- and LPS-induced upregulation of fluorescence intensity in HAECs (Fig. 5C and D). Moreover, LPS or Ox-LDL treatment for 3 h increased the mRNA levels of CCL2, ICAM1, VCAM1, SRLE, and IL6. Ad-MnSOD infection attenuated these effects in HAECs, except for VCAM1 and IL6 (Fig. 5E and I). Moreover, culture medium ELISA revealed that Ad-MnSOD infection reduced LPS or Ox-LDL-induced MCP-1 upregulation in HAECs (Fig. 5J). Western blot analysis revealed that Ad-MnSOD infection reduced LPS or Ox-LDL-induced ICAM-1 upregulation in HAECs (Fig. 5K and L).

Finally, we investigated the involvement of mtROS in hyperglycemia-induced atherogenic effects. Compared to normal glucose conditions, incubation with high glucose (25 mM) for 3 h, 24 h, or 7 days increased fluorescence intensity in HAECs (Fig. 6A). However, the high-glucose-induced increases in mRNA expression levels of CCL2, ICAM1, and VCAM1 were observed after 7 days of high-glucose incubation (Fig. 6B–F). In contrast, compared to the Ad-lacZ, infection with the Ad-MnSOD reduced high glucose-induced upregulation of fluorescence intensity in HAECs (Fig. 6G and H). Additionally, treatment with high glucose for 7 days increased the mRNA levels of CCL2, ICAM1, and VCAM1, whereas the mRNA levels of SELE and IL6 remained unchanged (Fig. 6I–M). Infection with the Ad-MnSOD, which continued the overexpression of MnSOD for 7 days in HAECs (Supplementary Fig. S4A and S4B), attenuated these effects (Fig. 6I–M). Moreover, culture medium ELISA revealed that Ad-MnSOD infection reduced high glucose-induced MCP-1 upregulation in HAECs (Fig. 6N). Western blot analysis revealed that Ad-MnSOD infection reduced high glucose-induced ICAM-1 upregulation in HAECs (Fig. 6O).

FIG. 5.

MnSOD overexpression suppresses mitochondrial ROS production and atherosclerosis-related molecules induced by Ox-LDL or LPS in HAECs. HAECs were infected with MnSOD-overexpressing (Ad-MnSOD) or control (Ad-lacZ) adenovirus for 48 h prior to the experiment. (A and B) MnSOD expression levels in HAECs were determined using Western blotting. Values were normalized to β-actin, and data were presented as the mean ± SEM from three independent experiments. Statistical analyses were performed using an unpaired t-test.*p < 0.05. (C and D) Adenovirus-infected HAECs were incubated with 20 µg/mL Ox-LDL or 10 ng/mL LPS for 3 h, and treated with 5 µM MitoSOX Red(red) for 20 min. Data were presented as the mean ± SEM from four independent experiments conducted in triple form. Statistical analyses were performed using one-way analysis of variance followed by Tukey’s multiple comparison test.*p < 0.05. (E–I) Adenovirus-infected HAECs were incubated with 20 µg/mL Ox-LDL or 10 ng/mL LPS for 3 h. The mRNA expression levels of each molecule were determined by real-time PCR. Values were normalized to Actb, and data were presented as the mean ± SEM from six independent experiments. Statistical analyses were performed using one-way analysis of variance followed by Tukey’s multiple comparison test.*p < 0.05. (J) Adenovirus-infected HAECs were incubated with 20 µg/mL Ox-LDL or 10 ng/mL LPS for 6 h. The MCP-1 concentration in the culture supernatants was measured by ELISA. Data were presented as the mean ± SEM from four independent experiments. Statistical analyses were performed using one-way analysis of variance followed by Tukey’s multiple comparison test.*p < 0.05. (K and L) Adenovirus-infected HAECs were incubated with 20 µg/mL Ox-LDL (K) or 10 ng/mL LPS (L) for 24 h. ICAM-1 protein expression was determined by Western blot analysis. Values were normalized to β-actin, and data were presented as the mean ± SEM five four independent experiments. Statistical analyses were performed using one-way analysis of variance followed by Tukey’s multiple comparison test.*p < 0.05.

FIG. 6.

MnSOD overexpression suppresses mitochondrial ROS production and atherosclerosis-related molecules induced by high glucose condition in HAECs. (A) HAECs were cultured under normal (5.5 mM) or high (25 mM) glucose conditions for 3, 24, or 168 h (7 days), followed by incubation with 5 µM MitoSOX Red(red) for 20 min. (B–F) HAECs were cultured under normal (5.5 mM) or high (25 mM) glucose conditions for 3, 24, or 168 h (7 days), and the mRNA expression of MCP-1 (B), ICAM-1 (C), VCAM-1 (D),E-selectin (E), and IL-6 (F) was determined using real-time PCR. Values were normalized to β-actin, and data were presented as the mean ± SEM from six independent experiments. *p < 0.05. (G–H) HAECs were infected with MnSOD-overexpressing (Ad-MnSOD) or control (Ad-lacZ) adenovirus for 48 h, and then cultured under normal (5.5 mM) or high (25 mM) glucose conditions for 3, 24, or 168 h (7 days), followed by incubation with 5 µM MitoSOX Red for 20 min. Data were presented as the mean ± SEM from four independent experiments conducted in triple form. Statistical analyses were performed using one-way analysis of variance followed by Tukey’s multiple comparison test. *p < 0.05. (I–M) HAECs were infected with Ad-MnSOD or Ad- lacZ for 48 h, and then cultured under normal glucose (5.5 mM) or high glucose (25 mM) conditions for 168 h (7 days). The mRNA expression levels of each molecule were determined by real-time PCR. Values were normalized to β-actin, and data were presented as the mean ± SEM from six independent experiments. Statistical analyses were performed using one-way analysis of variance followed by Tukey’s multiple comparison test. *p < 0.05. (N) HAECs were infected with Ad-MnSOD or Ad- lacZ for 48 h, and then cultured under normal (NG: 5.5 mM) or high (HG: 25 mM) glucose conditions for 168 h (7 days). The MCP-1 concentration in the culture supernatants was measured using ELISA. Data were presented as the mean ± SEM from four independent experiments. Statistical analyses were performed using one-way analysis of variance followed by Tukey’s multiple comparison test. *p < 0.05. (O) HAECs were infected with Ad-MnSOD or Ad- lacZ for 48 h, and then cultured under NG or HG conditions for 168 h (7 days). ICAM-1 protein expression was determined by Western blot analysis. Values were normalized to β-actin, and data were presented as the mean ± SEM for five independent experiments. Statistical analyses were performed using one-way analysis of variance followed by Tukey’s multiple comparison test. *p < 0.05.

Discussion

Previous reports that evaluated the effects of MnSOD heterozygous deficiency in ApoE KO mice fed a western diet (4% fat diet, Ballinger et al., 2002; Vendrov et al., 2017) consistently show that ROS in the vascular wall as well as the area of atherosclerotic lesions increase, suggesting that the decrease in MnSOD expression and the resulting increase in mtROS may be involved in atherosclerosis progression. In this study, we found that MnSOD overexpression, specifically in vascular endothelial cells in HFD-fed ApoE KO mice, regulates ROS in atherosclerotic lesions and can inhibit atherosclerosis by approximately 47% in the thoracic aorta and approximately 35% in the valve ring without directly affecting body weight, glucose metabolism, or lipid metabolism. Notably, additional histological analyses revealed that MnSOD overexpression also altered plaque composition. In contrast, despite MnSOD overexpression in the vascular endothelium, NCD-fed ApoE KO mice did not show a significant inhibitory effect. However, plaque formation tended to decrease, suggesting that mtROS regulation in vascular endothelial cells may strongly indicate atherosclerotic inhibition, which occurs only when an HFD is consumed.

In addition to chronic elevation of blood glucose levels, the NCD and HFD groups may have had other factors that increased mtROS production in vascular endothelial cells, such as hyperinsulinemia and increased LDL, a subsequent increased in Ox-LDL due to obesity, and increased serum LPS levels due to changes in the intestinal bacterial environment (Kim et al., 2012). In fact, in this study, the HFD group showed increased blood glucose levels, weight gain, and insulin resistance, as well as increased blood insulin levels, increased LDL cholesterol, and increased serum LPS compared to those in the NCD group. Furthermore, increases in relative mtROS levels has been induced in bovine aortic endothelial cells using hyperglycemic stimulation (Sada et al., 2016) as well as in human pulmonary microvascular and vascular endothelial cells using LPS stimulation (Wang et al., 2021; Zhu et al., 2023). Finally, mtROS production has been induced using Ox-LDL in mouse peritoneal macrophages (Chen et al., 2019). Using HAECs, we investigated the effects of mtROS production under various conditions and the effects on vascular endothelial cells regarding atherosclerosis progression. Although there was a slight time lag between each stimulus, Ox-LDL, LPS, and high blood glucose directly increased relative mtROS levels, suggesting that these factors may be involved in mtROS production. In contrast, ROS is induced by insulin in HepG2 cells (Iqbal et al., 2013) and mouse myoblasts (Loh et al., 2009); however, by using HAECs, insulin stimulation at concentrations that would be expected in clinical practice did not increase relative mtROS levels, suggesting that hyperinsulinemia may not be a trigger for mtROS production in vascular endothelial cells.

Changes in vascular reactivity cause damage to vascular endothelial cells and other vascular component cells, leading to atherosclerosis (Ross, 1993). This hypothesis proposes that atherosclerosis is caused by proliferative and organized lesions of the vascular wall associated with inflammatory responses due to vascular injury in the early stages of the disease. In the present study, analysis of the aortic valve rings of mice showed that MnSOD overexpression suppressed the intensity of 8-OHdG staining in atherosclerotic lesion sites. Simultaneously, the number of F4/80-positive cells, which are thought to be macrophages that have gathered at the atherosclerotic lesion site, was also suppressed by MnSOD overexpression. MnSOD overexpression significantly suppressed the mRNA expression of MCP-1, which is involved in monocyte migration, and ICAM-1, which is thought to be involved in macrophage adhesion to the endothelium, in the mouse aorta. Accordingly, the decrease in the number of macrophages in the aortic plaques of MnSOD overexpressing mice is due to the suppression of the expression of these molecules involved in monocyte migration and adhesion.

Several epidemiological studies, including familial hypercholesterolemia, have shown that an increase in LDL is an important risk factor for atherosclerosis (Boren et al., 2020; Libby, 2021). Furthermore, blood LDL infiltrating the vascular wall via vascular endothelial cells becomes Ox-LDL due to the addition of oxidative stress, exerting a powerful atherosclerotic effect (Goyal et al., 2012). In contrast, sdLDL increases in pathological conditions with an increase in remnant lipoproteins, such as in this model mouse (Higashioka et al., 2021; Véniant et al., 2001), and sdLDL is more susceptible to oxidative modification than normal LDL (Tribble et al., 2001). In this study, we also confirmed that sdLDL was significantly increased in the HFD group compared to the NCD group, suggesting that Ox-LDL may have triggered the promotion of atherosclerosis in the HFD group. Furthermore, Ox-LDL affects mitochondrial respiration in macrophages (Chen et al., 2019) and endothelial cells (Zmijewski et al., 2005) and increases relative mtROS levels; thus, Ox-LDL likely induces relative mtROS level upregulation in endothelial cells and affects atherosclerosis progression. Furthermore, Ox-LDL promotes endothelial cell activation by inducing the expression of adhesion molecules (Obermayer et al., 2018) and is involved in macrophage aggregation by inducing MCP-1 production (Wiesner et al., 2013) and other processes (Chen and Khismatullin, 2015). In this study, MCP-1, ICAM-1, and E-selectin production by Ox-LDL in HAECs was suppressed by MnSOD overexpression, suggesting that increased expression of these molecules via increased relative mtROS levels may be one of the mechanisms by which Ox-LDL promotes atherosclerosis.

LPS, an antigen that activates the immune system in animals and humans and is thought to play an important role in the pathogenesis of inflammatory diseases such as obesity, type 2 diabetes, and dyslipidemia, increases in the blood due to changes in the intestinal environment caused by an HFD (Kim et al., 2012). Furthermore, LPS stimulation increases mtROS in vascular endothelial cells (Zhu et al., 2023). In this study, we confirmed that the blood LPS concentration was higher in the HFD group than that in the NCD group, even in ApoE KO mice. LPS induces ROS production via NADPH oxidase in mouse microglia (Qin et al., 2004) and ROS production via Jun N-terminal kinase in human neutrophils (Khan et al., 2017). LPS induces mtROS in human pulmonary microvascular endothelial cells (Wang et al., 2021).

We showed that LPS also increases relative ROS levels derived from mitochondria, which is concordant with a similar result in a study using mouse microglial cells (Park et al., 2015). The key point of our research is that LPS-derived increase in relative mtROS levels can be suppressed by MnSOD overexpression, which may lead to the development of new treatments for various pathological conditions involving excessive ROS levels via mitochondrial dysfunction. LPS promotes inflammatory cytokine production via nuclear factor-Kappa-B (NF-κB) activation (Li et al., 2016). We have previously reported that excessive mtROS production under high glucose conditions is involved in vascular endothelial growth factor and cyclooxygenase-2 expression via the activation of nuclear factor kappa B (NF-κB) in studies using endothelial cells and mesangial cells (Nishikawa et al., 2000) (Kiritoshi et al., 2003). Accordingly, the increase in mtROS production induced by an HFD promotes atherosclerosis via NF-κB activation; however, further research is required to elucidate the mechanism of action.

The increase in relative mtROS levels in HAECs under hyperglycemic conditions was consistent with previous reports using bovine aorta endothelial cells, human retinal vascular endothelial cells, and mesangial cells (Brownlee, 2001; Fujisawa et al., 2009; Sada et al., 2016). Previous studies on the mechanism of increased mtROS due to hyperglycemia have suggested that the free NADH/NAD+ ratio in the cytoplasm induced by hyperglycemia may be a factor, as observed with hyperglycemic pseudohypoxia (Sada et al., 2016). In addition, an increase in the NADH/NAD+ ratio is a symptom characteristic of patients with poor diabetes control (Williamson et al., 1993). An increase in the NADH/NAD+ ratio plays an important role in the development of diabetic complications (Sada et al., 2016). In this study, we observed that high blood glucose stimulation for a short period increased relative mtROS levels, which was maintained for 7 days. However, an increase in atherosclerosis-related molecules was not observed with short-term stimulation. However, long-term high blood glucose stimulation for 7 days induced atherosclerosis-related molecule expression. The time lag between increase in relative mtROS levels and atherosclerosis-related molecule expression remains unknown. However, we hypothesize that the expression of atherosclerosis-related factors under hyperglycemic stimulation requires not only mtROS but also the involvement of additional factors. To confirm this hypothesis, further studies are needed. However, elevated blood glucose levels may also affect atherosclerosis progression in mice fed an HFD via mtROS production.

The potential influence of blood pressure on atherosclerotic lesion development should be considered. HFD feeding increases blood pressure in mice (Man et al., 2023 ), raising the possibility that differences in systemic hemodynamics could indirectly affect atherosclerosis. To address this concern in our experimental model, we performed additional measurements of systolic blood pressure in the key groups. Our data demonstrated no significant differences in blood pressure between eMnSOD-Tg and control ApoE KO mice, including after HFD feeding. These findings suggest that the reduced atherosclerotic lesion formation observed in eMnSOD-Tg mice is unlikely to be mediated by systemic blood pressure changes, but rather reflects direct effects of endothelial MnSOD overexpression on vascular redox regulation and atherogenesis.

Several large-scale randomized clinical trials have examined whether antioxidant therapies reduce cardiovascular events, but the results have been consistently disappointing. Supplementation with non-specific antioxidants, including vitamins E and C or β-carotene, failed to reduce major cardiovascular outcomes in diverse high-risk populations, as demonstrated in the HOPE study (Yusuf et al., 2000), Heart Protection Study (Heart Protection StudyCollaborative Group, 2002), Alpha-Tocopherol Beta-Carotene Cancer Prevention (ATBC) Study (The Alpha-Tocopherol, BetaCarotene Cancer Prevention Study Group, 1994), Physicians’ Health Study II (Sesso et al., 2008), and Women’s Antioxidant Cardiovascular Study (Cook et al., 2007). In some settings, these interventions were associated with adverse outcomes. These findings indicate that systemic antioxidant supplementation does not provide cardiovascular protection and may interfere with physiological redox signaling. In contrast, the CANTOS trial (Ridker et al., 2017) provided compelling evidence that targeting inflammation, specifically through interleukin-1β pathway inhibition, reduces recurrent cardiovascular events independently of lipid lowering. Although CANTOS did not directly implicate ROS as a therapeutic target, accumulating experimental evidence suggests that mitochondrial ROS act upstream of inflammatory activation, including inflammasome signaling and macrophage polarization. From this perspective, the success of anti-inflammatory therapy does not negate a pathogenic role for ROS, but rather suggests that indiscriminate scavenging of ROS may be an ineffective strategy to modulate disease-driving redox pathways.

Notably, mitochondrial redox regulation mediated by MnSOD represents a fundamentally different biological mechanism from exogenous antioxidant supplementation. MnSOD is an endogenous mitochondrial enzyme that selectively detoxifies superoxide within the mitochondrial matrix, thereby preserving redox homeostasis while maintaining physiological ROS signaling. Our findings therefore provide a mechanistically distinct framework in which endothelial cell-specific modulation of mitochondrial superoxide influences atherosclerosis progression, particularly under conditions of metabolic stress such as HFD-induced obesity. This concept complements, rather than contradicts, the clinical evidence from CANTOS and prior antioxidant trials, and highlights the importance of compartment- and cell-specific redox regulation in cardiovascular disease.

This study has several limitations. First, although MnSOD functions as a mitochondrial superoxide scavenger, we did not use the gold-standard assay for mitochondrial superoxide, namely HPLC-based quantification of the specific oxidation product mito-2-hydroxyethidium. Instead, we evaluated mitochondrial superoxide using MitoSOX Red, which provides greater specificity than general ROS indicators and yielded results consistent with the original observations. Nevertheless, the absence of HPLC analysis remains an important limitation. Second, all in vivo experiments were performed exclusively in male ApoE KO mice. Sex-dependent differences in atherosclerosis have been reported in ApoE KO mice. For example, Yi et al. demonstrated that partial deficiency of lipoic acid synthase exacerbated atherosclerotic lesion formation in male, but not in female, ApoE KO mice, indicating that biological sex can modify atherogenic responses even within the same genetic background (Yi et al., 2010). Therefore, the use of only male mice in the present study limits our ability to assess potential sex-dependent effects of MnSOD-related redox regulation during atherogenesis. Future studies including both sexes will be required to clarify these sex-specific differences. Third, although we performed additional histological analyses demonstrating reduced necrotic core formation and increased fibrotic content within atherosclerotic plaques of eMnSOD-Tg/ApoE KO mice, the present study did not directly assess vascular function or plaque rupture events. Although necrotic core size and collagen content are widely accepted surrogate markers of plaque stability in murine models of atherosclerosis, they do not fully capture the dynamic mechanical and functional properties of the vessel wall. Therefore, the absence of direct measurements of vascular reactivity, hemodynamic parameters, or spontaneous plaque rupture limits the extent to which our findings can be extrapolated to plaque stability and clinical cardiovascular events. Further studies incorporating functional vascular assessments and advanced analyses of plaque vulnerability will be required to define the role of endothelial MnSOD-mediated redox regulation more comprehensively in atherosclerotic disease progression. Finally, the enzymatic activity of MnSOD was not directly measured, despite the observed increase in endothelial MnSOD protein expression and reduction in mitochondrial superoxide levels in eMnSOD-Tg/ApoE KO mice. Emerging evidence indicates that MnSOD activity is regulated not only by protein abundance but also by post-translational modifications, particularly acetylation at lysine 68 (K68). HFD–induced oxidative stress has been associated with increased acetylation of MnSOD at K68, which can be prevented by sirtuin 3 (SIRT3) overexpression (Zhang et al., 2015). Moreover, vascular dysfunction has been linked to SIRT3 deficiency and increased MnSOD-K68 acetylation without significant changes in total MnSOD protein levels (Dikalova et al., 2017), highlighting the functional importance of MnSOD acetylation status in regulating mitochondrial redox balance. Notably, mice expressing a deacetylation-mimetic MnSOD mutant (K68R) are protected against oxidative stress and endothelial dysfunction (Dikalova et al., 2024). These findings suggest that MnSOD activity is dynamically controlled by acetylation-dependent mechanisms in addition to protein expression. Therefore, future studies assessing MnSOD enzymatic activity and acetylation status will be important to further clarify the regulation of mitochondrial redox signaling in endothelial cells. Therapeutic strategies targeting both MnSOD expression and deacetylation may represent a promising approach to attenuate oxidative stress and vascular dysfunction in obesity-associated atherosclerosis.

In conclusion, MnSOD overexpression decreases relative mtROS levels in vascular endothelial cells induced by Ox-LDL, LPS, and high glucose. Furthermore, MnSOD overexpression inhibits atherosclerosis-related factor expression, such as MCP-1 and ICAM-1. Finally, MnSOD overexpression in vascular endothelial cells significantly suppresses atherosclerosis progression without affecting blood glucose levels or serum lipids in HFD-fed ApoE KO mice. This study shows the involvement of mtROS in atherosclerosis progression in obesity and MS. Controlling mtROS may be a new means of preventing atherosclerosis progression.

Materials and Methods

Materials

LPS from Escherichia coli O111:B4 was purchased from Wako Chemicals (Tokyo, Japan). Human insulin solution (catalog number 19278) was purchased from Sigma-Aldrich (Oakville, ON, Canada).

Transgenic mice generation

Endothelial cell-specific MnSOD transgenic (eMnSOD-Tg) mice were generated as previously described (Goto et al., 2008). Briefly, a Tie2-MnSOD transgene was constructed by placing human MnSOD cDNA under the control of the mouse Tie2 promoter and enhancer. The purified Tie2-MnSOD fragment was microinjected into C57BL/6 pronuclei to establish the transgenic line. Genotyping was performed using PCR on tail DNA with primers specific for the human MnSOD transgene (forward: 5'-CGACCTGCCCTACGACTACG-3'; reverse: 5'-TGACCACCACCATTGAACTT-3'). Heterozygous transgenic mice were maintained, and non-transgenic littermates served as controls. In our previous report (Goto et al., 2008), we demonstrated enhanced MnSOD immunofluorescence signals in vascular endothelial cells of the transgenic mice, confirming endothelial cell-specific MnSOD overexpression. Although semiquantitative, these findings support robust endothelial MnSOD overexpression in eMnSOD-Tg mice.

Animal experiments

eMnSOD-Tg mice with a C57BL/6 background were generated as described previously (Goto et al., 2008). We obtained apolipoprotein E-knockout (ApoE KO) mice (C57BL/6 background) from Jackson Laboratories (Bar Harbor, ME). eMnSOD-Tg mice were crossbred with (ApoE KO) mice to obtain ApoE KO, eMnSOD-Tg mice.

To evaluate MnSOD expression, we performed western blotting using protein extracts from the thoracoabdominal aorta. MnSOD expression was approximately 2.6-fold higher in eMnSOD-Tg mice than in control littermates (Supplemental Fig. 5A and 5B).

Male mice were housed at 20–22°C on a 12 h light/dark cycle with an average of 50% humidity and fed either an NCD (#Rodent Diet CE-2, CLEA, Tokyo, Japan) or an HFD (60% calories from fat; #HFD32, CLEA, Tokyo, Japan) in the animal facility at Kumamoto University, Japan. All experiments with research animals were performed in accordance with institutional ethical guidelines and approved by the licensing committee of Kumamoto University (approval numbers: A2021-091 and A2023-042).

At 12 weeks of age, all male mice were fed either an NCD or HFD. Body weight, food intake, and casual blood glucose levels were measured at least once a week. After 8 weeks, the mice were administered intraperitoneal glucose tolerance (injected glucose: 1.5 g/kg) and intraperitoneal insulin tolerance tests (injected insulin: 1 U/kg). Mice were euthanized under general anesthesia with isoflurane to collect serum and aorta samples. Six-micrometer-thick frozen sections of aortic sinuses were stained with Oil Red O (Matsumura et al., 2011). Digital microphotographs of the aortic sinus were analyzed for lesion sizes in specific regions by measuring the stained surface area using a BZ-X700 microscope (Keyence, Tokyo, Japa). Sections of the aortic valve annulus were stained with a hematoxylin and eosin (H&E) kit (ab245880, Abcam, Cambridge, UK). Necrotic core areas were quantified using ImageJ software. Collagen fiber area in each section was quantified from Masson's trichrome-stained images (HT15 Trichrome Stain Kit, Sigma-Aldrich, St. Louis, MO, USA) using ImageJ software. Plasma total cholesterol, low-density lipoprotein cholesterol (LDL-C), triglyceride, high-density lipoprotein cholesterol, chylomicron cholesterol, very low-density lipoprotein cholesterol, and sdLDL-C concentrations were measured at Skylight Biotech Inc. (Akita, Japan) (Matsumura et al., 2011).

Immunofluorescence

Six-micrometer-thick frozen sections of the aortic sinus were subjected to immunofluorescence staining using primary antibodies against F4/80 (1:100; Abcam, Cambridge, UK) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) (1:100; Novus Biologicals, Centennial, USA). Secondary antibodies conjugated with Alexa Fluor 594 and Alexa Fluor 488 (1:100; Thermo Fisher Scientific, Tokyo, Japan) were used for detection. Fluorescent images were captured using a BZ-X700 microscope (Keyence, Tokyo, Japan).

Serum LPS levels

Serum LPS concentrations were quantified using the ToxinSensor^™ Chromogenic LAL Endotoxin Assay Kit (catalog #L00350; GenScript USA Inc., Piscataway, NJ) according to the manufacturers’ instructions.

Cell culture

HAECs (male) were purchased from PromoCell company (Heidelberg, Germany) and used in passages 4–10. Cells were cultured in an endothelial cell basal medium (Takara et al., Japan) with 5% fetal bovine serum and antibiotic-antimycotic mixed stock solution (Nacalai Tesque, Kyoto, Japan). Cells were maintained at 37°C in 5% CO₂ and 95% air and cultured according to the manufacturer’s instructions. The cells were also cultured for the appropriate time under various stimulation conditions.

Lipoprotein preparation

Human LDL (d = 1.019–1.063 g/mL) was isolated by ultracentrifugation from plasma samples obtained from consenting normolipidemic subjects after an overnight fast (Matsumura et al., 1997). LDL was dialyzed against 0.15 M NaCl and 1 mM ethylenediaminetetraacetic acid (EDTA) (pH 7.4). Ox-LDL was prepared by incubating LDL with 5 μM CuSO₄ for 20 h at 37°C, followed by adding 1 mM EDTA and cooling (Matsumura et al., 1997). The BCA protein assay reagent (Pierce) was used to determined the protein concentrations. The endotoxin level in the prepared Ox-LDL was <1 pg/μg protein, as measured using a Toxicolor System (Seikagaku Corp., Tokyo, Japan) (Matsumura et al., 1997).

Adenoviral vectors

Human manganese superoxide dismutase (MnSOD; a specific SOD for mtROS) adenoviral vectors (Ad-MnSOD) and lacZ control adenovirus (Ad-lacZ) were provided by Dr. Michael Brownlee (Albert Einstein College of Medicine, Bronx, NY). Cells were infected with Ad-MnSOD 48 h before the experiments. MnSOD overexpression in HAECs was confirmed using Western blotting.

mtROS generation in vitro

To evaluate direct mtROS generation, we used the reduced MitoTracker Red CM-H2Xros probe (Thermo Fisher Scientific, MA), which specifically detects ROS generation in mitochondria (Kajihara et al., 2017; Kukidome et al., 2006; Yoshinaga et al., 2021). Briefly, HAECs were cultured under experimental conditions and incubated with 300 nM CM-H2XRos at 37°C for 30 min before the end of the experiment. Cells were fixed with 4.0% paraformaldehyde (WaKo) for 15 min at room temperature.

Western blotting

Western blotting was performed on extracts of HAECs treated for 24 h or 168 h, and lysed in Cell Lysis Buffer (Cell Signaling Technology, Danvers, MA, USA) supplemented with protease and phosphatase inhibitors. Equal amounts of protein were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. The membranes were probed with purified primary antibodies using ICAM-1, MnSOD and β-Actin mouse monoclonal antibody (1:1000; Santa Cruz Biotechnology Inc, Dallas, Texas, USA.). After incubation with horse raddish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:1000, No. SA00001-1, Proteintech) as the secondary antibody, the proteins were visualized using the Immobilon Western Chemiluminescent HRP Substrate (Millipore, MA) according to the manufacturer’s instructions. Signals were quantified using a LuminoGraph I CMOS (ATTO, Tokyo, Japan). The intensity of the bands was quantified with ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Real-time quantitative reverse transcription–polymerase chain reaction (PCR)

Total cellular RNA from HAECs and mouse whole aortas was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany). First-strand complementary DNAs were synthesized using ReverTra Ace qPCR RT Master Mix (Toyobo). PCR was performed using the GoTaq qPCR Master Mix Protocol (Promega, CA) in VIIA7 Real-Time PCR System (Applied Biosystems, Foster City, CA). The sequences of primers used to amplify mouse and human Mcp1 (MCP1), Icam1 (ICAM1), Vcam1 (VCAM1), Sele (SELE), Il6 (IL6), and Actb (ACTB; β-actin) are listed in Supplementary Table S1. The expression levels of these genes were normalized to the Actb (ACTB) level in both mice and humans. To assess the specificity of the amplified PCR products, a dissociation curve analysis was performed after the last amplification cycle.

Enzyme-linked immunosorbent assay

Cell culture supernatant MCP-1 content was measured using ELISA (MCP-1, ab179886, Abcam). Fasting serum insulin was measured using the mouse insulin ELISA kit from FUJIFILM Wako (Osaka, Japan). These assays were performed according to the manufacturer’s protocol, and data were detected using a plate reader.

Statistics

All data are expressed as the mean ± standard error of the mean (SEM). Statistical analyses were performed using unpaired Student’s t-test for two-group comparisons and one-way analysis of variance, followed by Tukey’s multiple comparison test for more than two groups using GraphPad Prism software (ver. 10.4.1; GraphPad Software, Boston, MA). Prior to statistical comparisons, the normality of the data distribution was assessed using the D’Agostino–Pearson normality test and by visual inspection of Q–Q plots. Parametric statistical analyses were performed only after confirmation that the data did not significantly deviate from a normal distribution.

Authors’ Contributions

T.Y. and T.M. designed and conducted the experiments, analyzed the data, and drafted the article. N.K., Y.Y., Y.Z., M.T., and T.U. performed the experiments and contributed to data analysis. K.F., D.K., H.M., T.S., M.H., T.K., and T.N. interpreted the data and contributed to article preparation. E.A. and N.K. supervised the entire project. All authors read and approved the final version of the article.

Footnotes

Acknowledgments

The authors appreciate the advice and assistance from members of Center for Animal Resources and Development (CARD) at Kumamoto University. We would like to thank Editage () for English language editing.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

This study was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science, Japan (No. 23K07533 to Takeshi Matsumura, No. 22K15687 to Nobuhiro Kajihara, and 24K19003 to Saiko Nishida).

Supplemental Material

Abbreviations Used

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