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. 2023 Mar;39(2):309–318. doi: 10.6515/ACS.202303_39(2).20221027A

The Roles of Aerobic Exercise and Folate Supplementation in Hyperhomocysteinemia-Accelerated Atherosclerosis

Xingming Zhong 1, Rong He 2*, Shaohua You 1, Bo Liu 3, Xiujie Wang 1, Jieming Mao 4
PMCID: PMC9999187  PMID: 36911543

Abstract

Background

Hyperhomocysteinemia (HHcy) is an independent risk factor for atherosclerosis. Effective interventions to reduce HHcy-accelerated atherosclerosis are required.

Objectives

This study aimed to investigate the effects of aerobic exercise (AE) and folate (FA) supplementation on plasma homocysteine (Hcy) level and atherosclerosis development in a mouse model.

Methods

Six-week-old female apoE-/- mice were grouped into five groups (N = 6-8): HHcy (1.8 g/L DL-homocysteine (DL-Hcy) in drinking water), HHcy + AE (1.8 g/L DL-Hcy and aerobic exercise training on a treadmill), HHcy + FA (1.8 g/L DL-Hcy and 0.006% folate in diet), HHcy + AE + FA (1.8 g/L DL-Hcy, 0.006% folate, and aerobic exercise training on a treadmill), and a control group (regular water and diet). All treatment was sustained for 8 weeks. Triglyceride, cholesterol, lipoprotein, and Hcy levels were determined enzymatically. Plaque and monocyte chemoattractant protein-1 (MCP-1) expression levels in mouse aortic roots were evaluated by immunohistochemistry.

Results

Compared to the HHcy group (18.88 ± 6.13 μmol/L), plasma Hcy concentration was significantly reduced in the HHcy + AE (14.79 ± 3.05 μmol/L, p = 0.04), HHcy + FA (9.4 ± 3.85 μmol/L, p < 0.001), and HHcy + AE + FA (9.33 ± 2.21 μmol/L, p < 0.001) groups. Significantly decreased aortic root plaque area and plaque burden were found in the HHcy + AE and HHcy + AE + FA groups compared to those in the HHcy group (both p < 0.05). Plasma MCP-1 level and MCP-1 expression in atherosclerotic lesions were significantly decreased in the HHcy + AE and HHcy + AE + FA groups compared to the HHcy group (all p < 0.05).

Conclusions

AE reduced atherosclerosis development in HHcy apoE-/- mice independently of reducing Hcy levels. FA supplementation decreased plasma Hcy levels without attenuating HHcy-accelerated atherosclerosis. AE and FA supplementation have distinct mechanisms in benefiting atherosclerosis.

Keywords: Aerobic exercise, Atherosclerosis, Folate, Homocysteine, Hyperhomocysteinemia

Abbreviations

ACS, Acute coronary syndrome

AE, Aerobic exercise

ANOVA, Analysis of variance

ApoE, Apolipoprotein E

BHMT, Betaine homocysteine S-methyltransferase

CAD, Coronary artery disease

CE, Cholesterol

Cys, Cysteine

DL-Hcy, DL-homocysteine

ELISA, Enzyme-linked immunosorbent assay

FA, Folate

GSH-Px, Glutathione peroxidase

Hcy, Homocysteine

HDL-c, HDL cholesterol

HHcy, Hyperhomocysteinemia

IACUC, Institutional Animal Care and Use Committee

IOD, Integrated optical density

JAK/STAT, Janus kinase/signal transducer and activator of transcription

LDL, Low-density lipoprotein

LDL-c, LDL cholesterol

Lp, Lipoprotein

LV, Left ventricle

MCP-1, Monocyte chemoattractant protein-1

Met, Methionine

MTHFR, 5,10-methyltetrahydrofolate reductase

SHR, Spontaneously hypertensive rats

SYD, Shen-Yuan-Dan

TC, Total cholesterol

TCM, Traditional Chinese medicine

TG, Triglyceride

INTRODUCTION

Atherosclerosis is chronic arterial inflammation caused by the deposition of low-density lipoprotein (LDL) in the arterial wall and vasculitis and plaque formation.1 Plaque occupies space in the arterial lumen and restricts blood flow, which causes chronic abiotrophy.2 Atherosclerosis is an underlying cause of about 50% of all deaths in westernized societies due to the frequent development into ischemic heart disease, acute myocardial infarction, and peripheral vascular disease.3,4 However, the symptoms of atherosclerosis are mild and hard to determine during disease progression.5 Accordingly, understanding the pathogenesis of atherosclerosis can help to prevent or treat it more appropriately.

Hyperhomocysteinemia (HHcy) has been positively correlated with the pathogenesis of atherosclerosis.6 HHcy is defined as a condition with serum homocysteine concentration higher than 30 μM (normal range between 5-15 μM).7 Homocysteine (Hcy) is involved in the methionine cycle and can be converted into cysteine by cystathionine lyase.8 Through inherited or nutritional reasons, serum Hcy increases and causes oxidative stress in the vascular wall.9,10 Increased oxidative stress and Hcy further recruit LDL and macrophages into the vascular wall, initiate inflammatory responses in the vascular wall, and finally form plaque.11-13 The serum Hcy level of patients with acute coronary syndrome is correlated with the severity of coronary artery disease.14 Therefore, avoiding HHcy may help prevent the pathogenesis of atherosclerosis.

HHcy has two causes: nutrient-mediated and inherited.9 Inherited HHcy, accounting for approximately 5% of cases, is caused by genetic disorder of the methionine cycle or folate metabolism, particularly 5,10-methyltetrahydrofolate reductase, the key enzyme in folate metabolism.15 Nutrient-mediated HHcy is closely associated with vitamin B6, B12, and folate deficiency, which blocks methionine recycling from Hcy and cysteine produced from Hcy.16,17 One study focusing on the relationship between HHcy and serum folate level showed positive correlations with each other.18 Additionally, aerobic exercise has been shown to ameliorate atherosclerosis by modulating vascular inflammation and also to reduce HHcy in some cases.19 These studies imply that dietary folate supplementation and aerobic exercise may be therapeutic options for HHcy. However, a clinical trial which gave folate to HHcy patients reported that only 40% of the patients exhibited an improvement, suggesting that the attenuation of HHcy by dietary folate supplementation is limited.20 Studies on the therapeutic effect of aerobic exercise on HHcy have reported controversial results of both constant and decreased amounts of Hcy after aerobic exercise.21-23 Apart from aerobic exercise and dietary folate supplementation, no other convincing options for treating HHcy have been proposed. That is, treatment options for HHcy is still an unmet medical need. To investigate this issue, this study aimed to evaluate the effects of aerobic exercise and folate supplementation on plasma Hcy level and atherosclerosis development in a rodent model.

METHODS

Reagents

DL-homocysteine (DL-Hcy) and folate were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Anti-monocyte chemoattractant protein-1 (MCP-1) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Oil red O was purchased from Amresco (Solon, OH, USA).

Experimental animals and HHcy induction

We designed a five-arm study to investigate the attenuating efficacy of aerobic exercising, dietary folate supplementation, and their combination on HHcy and also the severity of atherosclerosis by evaluating arterial plaques in each group. The study included a control group (without any treatment), high-HHcy group (HHcy without treatment), aerobic exercise group (HHcy + AE), folate group (HHcy + FA), and combined group (HHcy + AE + FA). HHcy induction followed a previously published protocol.24 The designation of the study protocol followed the 2nd edition of the Institutional Animal Care and Use Committee (IACUC), and the protocol was reviewed and approved by the Institutional Ethics Committee for Animal Experiments of Peking University Health Science Center.

Thirty-eight 6-week-old female apoE-/- mice were purchased from the Experimental Animal Department of Peking University Health Science Center and kept in a controlled environment (22 °C, 12/12 day/night cycle) with ad libitum feeding. After one week of adaptation, the mice were grouped into 5 groups: animal control (N = 6), vehicle control (N = 8), aerobic exercise (N = 8), folate (N = 8), and combined group (N = 8). All animals except the control group were fed with 1.8 g/L DL-Hcy in drinking water for 63 days (regular drinking water for the control group). Each mouse’s drinking volume was monitored daily, and the weight was measured once a week.

Aerobic exercise and dietary folate supplementation

On day 8, the mice were treated according to the study design: the animal control and vehicle control groups kept DL-Hcy and regular drinking water feeding, the AE group started exercising using a motorized rodent treadmill (described below), the FA group was fed with a 0.006% folate diet,25 and the combined group with both aerobic exercise and folate diet. All treatments were maintained for 8 weeks, and drinking water in each group was given equally during treatment. The dimension of the left ventricle of each mouse was measured by ultrasound in the 4th and 8th weeks of the treatment.

Aerobic exercise protocol

The aerobic exercise protocol for the mice followed the description in guidelines.26 Mice in the AE group were acclimatized with a motorized rodent treadmill (BCPT-98, Hangzhou, China) with an exercising program of 10 m/min for 10 min (day 1), 11 m/min for 20 min (day 2), 12 m/min for 30 min (day 3), 13 m/min for 40 min (day 4), and 14 m/min for 50 min (day 5), respectively. After two days of rest, the mice performed aerobic exercise at a speed of 15 m/min for 60 min, once a day, five days a week, for eight weeks. The angle of the treadmill was kept at 0°.

Mice sacrifice and serum biochemical evaluation

On day 63, the mice were sacrificed by CO2 anesthetization. Afterward, blood was collected immediately using PBS perfusion, heparin anticoagulation, and centrifugation at 1500 × g for 15 min to prepare plasma. Analysis of total cholesterol (TC), LDL cholesterol (LDL-c), HDL cholesterol (HDL-c), triglyceride (TG), and Hcy levels was performed using an automated biochemistry analytic system (Olympus AU5400, Tokyo, Japan). Plasma MCP-1 concentration was determined using an ELISA kit (R&D Systems MJE00B, Minneapolis, MN, USA), as described previously.27

Immunohistochemical analysis and plaque quantification

After perfusion, the mice were fixed by 4% paraformaldehyde perfusion followed by aortic tree collection. Aortic trees with a length of 350 μm were frozen-sectioned with a 7-μm interval and stained with oil red O (for arterial plaque) or anti-MCP-1 antibody (1:100, for arterial MCP-1 quantification), as described previously.28 The arterial plaque was photographed and quantified using Image-pro Plus V5.0 (Media Cybernetics, Rockville, MD, USA). The area of arterial plaque in each mouse was measured in six consecutive sections and the average was calculated. Then, the average area was converted into plaque burden by dividing the plaque area by lumen area.29 Semiquantitative analysis of the MCP-1 expression level in aortic root plaque was performed according to the mean integrated optical density (IOD) using Image-Pro Plus 6.0 software.

Statistical analysis

Data are presented as means ± SD. The unpaired Student’s t-test and one-way analysis of variance (ANOVA) coupled with the least significant difference test were used to analyze statistical significance. Data analysis was performed using the SPSS 19.0 software package (SPSS, Chicago, IL, USA), and a value of p < 0.05 was considered statistically significant.

RESULTS

AE, FA, and AE + FA ameliorated HHcy in mice but not serum lipid content

No significant differences among the groups were found in body weight throughout the whole experiment (Supplementary Figure 1) or left ventricle dimension in the 4th and 8th weeks of treatment (Supplementary Table 1). After eight weeks of DL-Hcy treatment, a significantly higher plasma Hcy concentration was detected in the HHcy group than in the control group (18.88 ± 6.13 μmol/L in HHcy vs. 8.25 ± 1.42 μmol/L in the controls, p < 0.001), indicating the successful induction of HHcy in the mice. Hcy concentrations were significantly lower in the HHcy + AE (14.79 ± 3.05 μmol/L, p = 0.040), HHcy + FA (9.40 ± 3.85 μmol/L, p < 0.001), and HHcy + AE + FA (9.33 ± 2.21 μmol/L, p < 0.001) groups than in the HHcy group (Table 1). the HHcy + FA and HHcy + AE + FA groups had comparable Hcy concentrations to the control group. Also, the Hcy concentration in the HHcy + FA group was similar to the HHcy + AE + FA group (p > 0.05, Table 1). All groups had comparable serum lipid concentrations (Table 1), indicating no dyslipidemia in the mice. These results revealed that FA and FA + AE were able to ameliorate diet-induced HHcy.

Supplementary Figure 1.

Supplementary Figure 1

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The weekly mean live body weight in all groups of apoE-/- mice. AE, aerobic exercise; FA, folate; HHcy, hyperhomocysteinemia.

Supplementary Table 1. The 4-week and 8-week left ventricle dimension in all apoE-/- mice groups.

  Control HHcy HHcy + AE HHcy + FA HHcy + AE + FA p value
LVIDd (mm) 4 week 2.89 ± 0.43 2.40 ± 0.49 2.61 ± 0.54 2.84 ± 0.51 2.54 ± 0.19 0.316
LVIDd (mm) 8 week 2.81 ± 0.26 2.42 ± 0.41 2.22 ± 0.29 2.38 ± 0.36 2.58 ± 0.61 0.150
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The HHcy groups of apoE-/- mice were fed high Hcy water for 8 weeks.

AE, aerobic exercise; FA, folic acid; HHcy, hyperhomocysteinemia; LVIDd, left ventricular internal dimension end diastole; SD, standard deviation. Values are mean ± SD.

Table 1. Plasma Hcy and lipid levels in all groups of apoE−/− mice.

  Control HHcy HHcy + AE HHcy + FA HHcy + AE + FA p value
Fluid consumption (ml/d) 3.42 ± 2.00 3.22 ± 1.64 3.53 ± 2.07 3.53 ± 2.40 3.65 ± 2.47 0.996
Hcy (μmol/L) 8.25 ± 1.42 18.88 ± 6.13* 14.79 ± 3.05*# 9.4 ± 3.85#† 9.33 ± 2.21#† < 0.001
T-CHO (mmol/L) 8.19 ± 1.52 10.82 ± 1.01 8.29 ± 3.90 10.42 ± 1.47 9.64 ± 1.23 0.125
LDL-c (mmol/L) 1.94 ± 0.58 1.70 ± 0.36 1.77 ± 0.27 2.19 ± 0.92 1.61 ± 0.48 0.329
HDL-c (mmol/L) 0.60 ± 0.02 0.42 ± 0.12 0.42 ± 0.25 0.45 ± 0.23 0.59 ± 0.18 0.359
TG (mmol/L) 1.01 ± 0.28 1.03 ± 0.33 1.01 ± 0.18 1.15 ± 0.45 1.20 ± 0.39 0.623
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The HHcy groups of apoE−/− mice were fed high Hcy water for 8 weeks.

AE, aerobic exercise; FA, folic acid; HDL-c, high density lipoprotein cholesterol; HHcy, hyperhomocysteinemia; LDL-c, low density lipoprotein cholesterol; T-CHO, total cholesterol; TG, triglycerides.

Values are mean ± SD. * p < 0.05 as compared with control group; # p < 0.05 as compared with HHcy group; † p < 0.05 as compared with HHcy + AE group.

Atherosclerotic plaque area and plaque burden

Atherosclerotic lesions were identified in aortic roots and the major arterial branches of the aortic arch in the HHcy group (Figures 1, 2A). The area of arterial plaque in aortic rings was significantly higher in the HHcy group than in the control group (× 10-4 mm2, 16.56 ± 5.02 vs. 6.68 ± 7.15, p = 0.007; Figure 3A), revealing that the Hcy-induced atherosclerosis model had successfully been constructed. Compared to the HHcy group, the HHcy + AE (6.57 ± 6.24 × 10-4 mm2, p = 0.003) and HHcy + AE + FA (9.63 ± 5.38 × 10-4 mm2, p = 0.031) groups had significantly lower areas of arterial plaque (Figure 3A). After converting arterial plaque into plaque burden (Figure 3B), both the HHcy + AE (0.08 ± 0.07, p < 0.001) and HHcy + AE + FA groups (0.12 ± 0.05, p = 0.005) had significantly lower plaque burden than the HHcy (0.22 ± 0.06) group. Also, the plaque burden in the HHcy + FA group was not significantly different compared to the HHcy group. These results indicated that AE and AE + FA reduced HHcy-induced atherosclerosis.

Figure 1.

Figure 1

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Atherosclerotic lesions of the aortic tree in all groups of apoE-/- mice. All apoE-/- mice were categorized into 5 groups: control group, hyperhomocysteinemia (HHcy) group, HHcy + aerobic exercise (AE) group, HHcy + folate (FA) group and HHcy + AE + FA group. The HHcy groups of apoE-/- mice were fed high Hcy water for 8 weeks. The AE groups were trained in a motorized rodent treadmill for 8 weeks (speed: 15 m/min, slope: 0°, 60 min/d, 5 d/wk). The FA groups of mice were fed a high-folate diet containing 0.006% folate. Mice were sacrificed, and the aortic tree was dissected in wax for oil red O staining.

Figure 2.

Figure 2

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Serial sections of the aortic roots were stained with oil red O (A) and monocyte chemoattractant protein-1 (MCP-1) (B). Representative histological data of oil red O and MCP-1 staining in aortic roots in different groups of apoE-/- mice (magnification ×50). Both signals were markedly increased in HHcy group compared with control group. When compared with HHcy group, both signals were reduced obviously in HHcy + AE group and HHcy + AE + FA group. However, neither oil red O straining nor MCP-1 straining was decreased markedly in HHcy + FA group compared with HHcy group. Abbreviations are in Figure 1.

Figure 3.

Figure 3

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Aortic root plaque area (A) and plaque burden (B) in all groups of apoE-/- mice. * p < 0.05 when compared with control group, # p < 0.05 when compared with HHcy group; & p < 0.05 when compared with HHcy + FA group. Abbreviations are in Figure 1.

Plasm MCP-1 levels and MCP-1 expression in atherosclerotic lesions

Several serum indicators are regarded as novel biomarkers of atherosclerosis, such as MCP-1.30 MCP-1 recruits macrophages and monocytes and induces inflammation in tissues.31 Although the plasma MCP-1 concentration has not been correlated with disease activity or prognosis,32 it is considered as an initial biomarker for atherosclerosis.

Aortic root sections of all groups of the apoE-/- mice were stained with oil red O and immunohistochemically stained with MCP-1 to determine lipid accumulation and MCP-1 expression in mouse atherosclerotic lesions. As shown in Figure 2, oil red O and MCP-1 staining indicated lipid accumulation and inflammation in the arterial plaque, the typical feature of atherosclerosis.33 Using IOD to semi-quantify the expression level of MCP-1, a significantly higher IOD in the HHcy group than in the control group was confirmed (39.02 ± 14.94 vs. 9.53 ± 8.41, p < 0.05; Figure 4, Supplementary Table 2). MCP-1- and oil red O-positive areas were lower in the HHcy + AE and HHcy + AE + FA groups than in the HHcy group (Figure 2). After semi-quantification by IOD, significantly decreased MCP-1 expression levels were found in the HHcy + AE (12.06 ± 9.79, p < 0.05) and HHcy + AE + FA (12.37 ± 7.33, p < 0.05) groups than in the HHcy (39.02 ± 14.94) group (Figure 4, Supplementary Table 2). Comparable results in the staining of oil red O and MCP-1 expression were found between the HHcy + FA group and HHcy group (Figures 2, 4; Supplementary Table 2).

Figure 4.

Figure 4

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Plasma MCP-1 levels in all groups of apoE-/- mice. * p < 0.05 when compared with control group; # p < 0.05 when compared with HHcy group. Abbreviations are in Figure 1.

Supplementary Table 2. MCP-1 expression in aortic root plaque in all groups of apoE-/- mice.

  Control (N = 6) HHcy (N = 3) HHcy + AE (N = 4) HHcy + FA (N = 5) HHcy + AE + FA (N = 5) p value
IOD (mean) 9.53 ± 8.41 39.02 ± 14.94* 12.06 ± 9.79#† 32.90 ± 23.66* 12.37 ± 7.33#† 0.018
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The HHcy groups of apoE-/- mice were fed high Hcy water for 8 weeks.

AE, aerobic exercise; FA, folic acid; IOD, integrated optical density; HHcy, hyperhomocysteinemia; MCP-1, monocyte chemoattractant protein-1.

Values are mean ± SD; * p < 0.05 when comparing with control group; # p < 0.05 when comparing with HHcy group; † p < 0.05 when comparing with HHcy + FA group.

ELISA showed a significantly higher MCP-1 level in the HHcy group than in the control group (pg/mL, 126.13 ± 64.48 vs. 69.33 ± 35.38, p = 0.019; Figure 5). In addition, the HHcy + AE (61.48 ± 35.25 pg/mL, p = 0.004) and HHcy + AE + FA (65.16 ± 30.27 pg/mL, p = 0.006) groups exhibited significantly lower plasma MCP-1 concentrations than the HHcy group (Figure 5). Moreover, a comparable plasma MCP-1 concentration was found between the HHcy + FA and HHcy groups (Figure 5).

Figure 5.

Figure 5

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MCP-1 expression level in aortic root plaque in all groups of apoE-/- mice. * p < 0.05 when comparing with control group; # p < 0.05 when comparing with HHcy group; & p < 0.05 when comparing with HHcy + FA group. IOD, integrated optical density. Abbreviations are in Figure 1.

DISCUSSION

The present study showed a distinct phenomenon in HHcy-mediated atherosclerosis, in that aerobic exercise ameliorated arterial plaque formation but did not attenuate HHcy. On the contrary, folate supplementation effectively decreased HHcy but had no significant effect on diminishing arterial plaque. AE/FA synergism reduced either serum Hcy content or arterial plaque, whereas further dose escalation was essential. These results indicate that the mechanism of aerobic exercise training and folate supplementation in benefiting atherosclerosis is distinct.

We observed that dietary folate supplementation could reduce serum Hcy content but not ameliorate arterial plaque in the HHcy mice model. This result is different to a previous study which showed that folate could delay arterial plaque in a high-fat diet-induced HHcy model.34 Comparing the experimental protocols between these two studies, HHcy induced by high dietary fat could be offset by dietary folate supplementation in a regular diet.34 Furthermore, dietary folate supplementation could alter JAK/STAT signaling in myelomonocytic cells and release inflammatory cytokines, which might augment arterial plaque formation.35 In summary, the difference in results in folate-mediated arterial plaque might be attributed to folate in a regular rodent diet. Replacing a regular diet with a low-folate diet might eliminate this error from regular feeding.

Unlike dietary folate supplementation, aerobic exercise did not ameliorate HHcy but ameliorated arterial plaque in the HHcy model. The therapeutic effect of aerobic exercise on HHcy is a controversial issue. Previous studies have revealed that aerobic exercise prevents folate-restricted-mediated HHcy via increased activity of betaine homocysteine S-methyltransferase in renal cells, which converts Hcy into methionine.21,36 However, Wang et al. and Deminice et al. reported a negative correlation between regular aerobic exercise and HHcy in hypertensive patients.37,38 Iglesias-Gutiérrez et al. further demonstrated that sedentary individuals and runners experience a transient increase in Hcy after acute exercise due to augmented oxidative status by inhibiting glutathione peroxidase.22 Hence, the characteristics of aerobic exercise in HHcy are still uncertain. Nevertheless, aerobic exercise can surely attenuate the symptoms of HHcy by reducing aortic endothelial oxidative injury, remodeling adverse muscle, and reducing inflammation.39-42 Aerobic exercise can reduce the risk of atherosclerosis by attenuating the inflammatory response and modulating dyslipidemia.43-45 Animal studies have suggested that aerobic exercise limits atherosclerosis through promoting autophagy,46,47 similar to the mechanism of a traditional Chinese medicine Shen-Yuan-Dan.48 Hence, the clinical benefits of aerobic exercise on atherosclerosis are undoubted, even though they may not be related to remission of HHcy. Our study revealed that aerobic exercise decreased Hcy in the mice model, providing evidence for this issue.

In this study, arterial plaque with lipid accumulation in mice without dyslipidemia was observed. HHcy was induced by oral Hcy supplementation rather than hypercholesteremia or hyperlipidemia, which may explain the presence of atherosclerosis in the mice without dyslipidemia. Although oral Hcy supplementation is a reliable model for HHcy and ApoE-/- mice are susceptible to dyslipidemia induction,49,50 the induction of atherosclerosis in ApoE-/- mice still needs a high-fat diet.51 Therefore, the mice model in this study is similar to inherited HHcy more than diet-induced HHcy. In order to mimic diet-induced HHcy, a high-fat diet could be combined with oral Hcy supplementation or folate ablation in HHcy induction, which could accelerate HHcy and atherosclerosis onset.52 Notably, lipid drops were also observed in the HHcy-induced arterial plaque even though dyslipidemia was absent. This phenomenon could be attributed to HHcy-induced dyslipidemia. Kapoor et al. reported that HHcy and dyslipidemia could be induced by methionine administration.53 The detailed mechanism between HHcy (or higher serum Hcy) and dyslipidemia is still unclear. Nevertheless, Obeid and Herrmann reported that S-adenosyl methionine, the precursor of Hcy, may be a key intermediator.54 In conclusion, the absence of dyslipidemia in the Hcy-induced ApoE-/- mice model was foreseeable, and the pathology appears to be more similar to inherited HHcy than diet-induced HHcy.

A high-intensity exercise protocol has been associated with cardiac hypertrophy in spontaneously hypertensive rats (SHR).55 However, SHR cardiomyocyte hypertrophy was not observed in a study with a moderate-intensity aerobic exercise protocol.56 In the current study, no significant differences in body weight throughout the training program or left ventricle dimension were observed between the AE and non-AE groups. In addition, no cardiac hypertrophy was found in the AE group. These findings indicate that our study involved a moderate-intensity aerobic exercise protocol which did not cause cardiac hypertrophy.

For future studies, adding a high-fat diet in HHcy induction is recommended to simulate diet-induced HHcy. Also, folate ablation and re-supplementation could verify the significance of folate in HHcy-induced atherosclerosis. For aerobic exercise/folate synergism, reducing dietary folate supplementation in a reduced folate diet could elucidate the potential of aerobic exercise/folate synergism.

The novel therapeutic efficacy of folate/aerobic exercise synergism on cardiac hypertrophy in this study is interesting. Previous studies have demonstrated that HHcy increases the risk of left ventricular hypertrophy by modulating energy metabolism in cardiomyocytes.57,58 Additionally, intense and regular aerobic exercise stimulates the proliferation of cardiomyocytes and improves cardiac function via benign cardiac hypertrophy.59,60 However, our study revealed a mitigating effect of aerobic exercise on HHcy. Therefore, the effect of aerobic exercise on cardiac hypertrophy is uncertain. Besides, folate supplementation is known to prevent cardiac dysfunction in both healthy and obese mice models by inhibiting HHcy-mediated myocardial fibrosis.61,62 In addition, folate supplementation diminished HHcy. Accordingly, the therapeutic efficacy of folate supplementation on cardiac hypertrophy is promising.

CONCLUSIONS

Dietary folate supplementation ameliorated HHcy, and aerobic exercise attenuated atherosclerosis development. Both have distinct mechanisms in benefiting atherosclerosis. Combining both efficiently attenuated HHcy and arterial plaque.

Acknowledgments

None.

DECLARATION OF CONFLICT OF INTEREST

All the authors declare no conflict of interest.

FUNDING

This study was funded by the Beijing Municipal Administration of Hospitals Incubation Program (grant number PX2019036).

REFERENCES

  • 1.Wu MY, Li CJ, Hou MF, Chu PY. New insights into the role of inflammation in the pathogenesis of atherosclerosis. Int J Mol Sci. 2017;18:2034. doi: 10.3390/ijms18102034. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Chernyavskiy I, Veeranki S, Sen U, Tyagi SC. Atherogenesis: hyperhomocysteinemia interactions with LDL, macrophage function, paraoxonase 1, and exercise. Ann N Y Aca Sci. 2016;1363:138–154. doi: 10.1111/nyas.13009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Aldons JL. Atherosclerosis. Nature. 2000;407:233–241. doi: 10.1038/35025203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Ala-Korpela M. The culprit is the carrier, not the loads: cholesterol, triglycerides and apolipoprotein B in atherosclerosis and coronary heart disease. Int J Epidemiol. 2019;48:1389–1392. doi: 10.1093/ije/dyz068. [DOI] [PubMed] [Google Scholar]
  • 5.Chen LX, Wang HH. Comparison between single- and double-bundle anterior cruciate ligament reconstructions for knee with grade 2 medial collateral ligament injury. Medicine. 2021;100:e24846. doi: 10.1097/MD.0000000000024846. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Chrysant SG, Chrysant GS. The current status of homocysteine as a risk factor for cardiovascular disease: a mini review. Expert Rev Cardiovasc Ther. 2018;16:559–565. doi: 10.1080/14779072.2018.1497974. [DOI] [PubMed] [Google Scholar]
  • 7.Ganguly P, Alam SF. Role of homocysteine in the development of cardiovascular disease. Nutr J. 2015;14:6. doi: 10.1186/1475-2891-14-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Chiocchetti A, Prodam F, Dianzani U. Homocysteine and folate in inflammatory bowel disease: can reducing sulfur reduce suffering? Dig Dis Sci. 2018;63:3161–3163. doi: 10.1007/s10620-018-5274-2. [DOI] [PubMed] [Google Scholar]
  • 9.Zaric BL, Obradovic M, Bajic V, et al. Homocysteine and hyperhomocysteinaemia. Curr Med Chem. 2019;26:2948–2961. doi: 10.2174/0929867325666180313105949. [DOI] [PubMed] [Google Scholar]
  • 10.Shemiakova T, Ivanova E, Grechko AV, et al. Mitochondrial dysfunction and DNA damage in the context of pathogenesis of atherosclerosis. Biomedicines. 2020;8:166. doi: 10.3390/biomedicines8060166. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Khatana C, Saini NK, Chakrabarti S, et al. Mechanistic insights into the oxidized low-density lipoprotein-induced atherosclerosis. Oxid Med Cell Longev. 2020;2020:5245308. doi: 10.1155/2020/5245308. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Li JJ, Li Q, Du HP, et al. Homocysteine triggers inflammatory responses in macrophages through inhibiting CSE-H2S signaling via DNA hypermethylation of CSE promoter. Int J Mol Sci. 2015;16:12560–12577. doi: 10.3390/ijms160612560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Batulan Z, Pulakazhi Venu VK, Li Y, et al. Extracellular release and signaling by heat shock protein 27: role in modifying vascular inflammation. Front Immunol. 2016;7:285. doi: 10.3389/fimmu.2016.00285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Karadeniz M, Sarak T, Duran M, et al. Hyperhomocysteinemia predicts the severity of coronary artery disease as determined by the SYNTAX score in patients with acute coronary syndrome. Acta Cardiol Sin. 2018;34:458–463. doi: 10.6515/ACS.201811_34(6).20180528B. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Brustolin S, Giugliani R, Felix TM. Genetics of homocysteine metabolism and associated disorders. Braz J Med Biol Res. 2010;43:1–7. doi: 10.1590/s0100-879x2009007500021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Ma Y, Peng D, Liu C, et al. Serum high concentrations of homocysteine and low levels of folic acid and vitamin B12 are significantly correlated with the categories of coronary artery diseases. BMC Cardiovasc Disord. 2017;17:37. doi: 10.1186/s12872-017-0475-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.McKinley MC, McNulty H, McPartlin J, et al. Low-dose vitamin B-6 effectively lowers fasting plasma homocysteine in healthy elderly persons who are folate and riboflavin replete. Am J Clin Nutr. 2001;73:759–764. doi: 10.1093/ajcn/73.4.759. [DOI] [PubMed] [Google Scholar]
  • 18.Onyemelukwe OU, Maiha BB. Hyperhomocysteinemia and folate levels in normal healthy Nigerians living in Zaria: subanalysis of ABU homocysteine cross-sectional survey. Ann Afr Med. 2019;18:143–152. doi: 10.4103/aam.aam_53_18. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Abd El-Kader SM, Al-Shreef FM, Al-Jiffri OH. Impact of aerobic exercise versus resisted exercise on endothelial activation markers and inflammatory cytokines among elderly. Afr Health Sci. 2019;19:2874–2880. doi: 10.4314/ahs.v19i4.9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Tian H, Tian D, Zhang C, et al. Efficacy of folic acid therapy in patients with hyperhomocysteinemia. J Am Coll Nutr. 2017;36:528–532. doi: 10.1080/07315724.2017.1330162. [DOI] [PubMed] [Google Scholar]
  • 21.Neuman JC, Albright KA, Schalinske KL. Exercise prevents hyperhomocysteinemia in a dietary folate-restricted mouse model. Nutr Res. 2013;33:487–493. doi: 10.1016/j.nutres.2013.04.008. [DOI] [PubMed] [Google Scholar]
  • 22.Iglesias-Gutierrez E, Garcia-Gonzalez A, Montero-Bravo A, et al. Exercise-induced hyperhomocysteinemia is not related to oxidative damage or impaired vascular function in Amateur middle-aged runners under controlled nutritional intake. Nutrients. 2021;13 doi: 10.3390/nu13093033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Dos Santos DP, Ribeiro DF, Frigoli GF, et al. Voluntary exercise attenuates hyperhomocysteinemia, but does not protect against hyperhomocysteinemia-induced testicular and epididymal disturbances. Reprod Sci. 2022;29:277–290. doi: 10.1007/s43032-021-00704-1. [DOI] [PubMed] [Google Scholar]
  • 24.Zhou J, Moller J, Danielsen CC, et al. Dietary supplementation with methionine and homocysteine promotes early atherosclerosis but not plaque rupture in ApoE-deficient mice. Arterioscler Thromb Vasc Biol. 2001;21:1470–1476. doi: 10.1161/hq0901.096582. [DOI] [PubMed] [Google Scholar]
  • 25.Li M, Chen J, Li YS, et al. Folic acid reduces chemokine MCP-1 release and expression in rats with hyperhomocystinemia. Cardiovasc Pathol. 2007;16:305–309. doi: 10.1016/j.carpath.2007.03.005. [DOI] [PubMed] [Google Scholar]
  • 26.Poole DC, Copp SW, Colburn TD, et al. Guidelines for animal exercise and training protocols for cardiovascular studies. Am J Physiol Heart Circ Physiol. 2020;318:H1100–H1138. doi: 10.1152/ajpheart.00697.2019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Zeng X, Dai J, Remick DG, Wang X. Homocysteine mediated expression and secretion of monocyte chemoattractant protein-1 and interleukin-8 in human monocytes. Circulation Res. 2003;93:311–320. doi: 10.1161/01.RES.0000087642.01082.E4. [DOI] [PubMed] [Google Scholar]
  • 28.Zhou J, Werstuck GH, Lhotak S, et al. Association of multiple cellular stress pathways with accelerated atherosclerosis in hyperhomocysteinemic apolipoprotein E-deficient mice. Circulation. 2004;110:207–213. doi: 10.1161/01.CIR.0000134487.51510.97. [DOI] [PubMed] [Google Scholar]
  • 29.Centa M, Ketelhuth DFJ, Malin S, Gistera A. Quantification of atherosclerosis in mice. J Vis Exp. 2019;148 doi: 10.3791/59828. [DOI] [PubMed] [Google Scholar]
  • 30.Lin J, Kakkar V, Lu X. Impact of MCP-1 in atherosclerosis. Curr Pharm Des. 2014;20:4580–4588. doi: 10.2174/1381612820666140522115801. [DOI] [PubMed] [Google Scholar]
  • 31.Deshmane SL, Kremlev S, Amini S, Sawaya BE. Monocyte chemoattractant protein-1 (MCP-1): an overview. J Interferon Cytokine Res. 2009;29:313–326. doi: 10.1089/jir.2008.0027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Ding D, Su D, Li X, et al. Serum levels of monocyte chemoattractant protein-1 and all-cause and cardiovascular mortality among patients with coronary artery disease. PLoS One. 2015;10:e0120633. doi: 10.1371/journal.pone.0120633. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Malekmohammad K, Bezsonov EE, Rafieian-Kopaei M. Role of lipid accumulation and inflammation in atherosclerosis: focus on molecular and cellular mechanisms. Front Cardiovasc Med. 2021;8:707529. doi: 10.3389/fcvm.2021.707529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Cui S, Li W, Lv X, et al. Folic acid supplementation delays atherosclerotic lesion development by modulating MCP1 and VEGF DNA methylation levels in vivo and in vitro. Int J Mol Sci. 2017;18:990. doi: 10.3390/ijms18050990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Ma J, Zhen X, Huang X, Jiang X. Folic acid supplementation repressed hypoxia-induced inflammatory response via ROS and JAK2/STAT3 pathway in human promyelomonocytic cells. Nutr Res. 2018;53:40–50. doi: 10.1016/j.nutres.2018.03.007. [DOI] [PubMed] [Google Scholar]
  • 36.Mohammadi HR, Khoshnam E, Jahromi MK, et al. The effect of 12-week of aerobic training on homocysteine, lipoprotein A and lipid profile levels in sedentary middle-aged men. Int J Prev Med. 2014;5:1060–1066. [PMC free article] [PubMed] [Google Scholar]
  • 37.Wang W, Li J, Ji P, et al. Association between regular aerobic exercise and hyperhomocysteine in hypertensive patients. Postgrad Med. 2020;132:458–464. doi: 10.1080/00325481.2020.1743114. [DOI] [PubMed] [Google Scholar]
  • 38.Deminice R, Ribeiro DF, Frajacomo FT. The effects of acute exercise and exercise training on plasma homocysteine: a meta-analysis. PLoS One. 2016;11:e0151653. doi: 10.1371/journal.pone.0151653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Iglesias-Gutierrez E, Egan B, Diaz-Martinez AE, et al. Transient increase in homocysteine but not hyperhomocysteinemia during acute exercise at different intensities in sedentary individuals. PLoS One. 2012;7:e51185. doi: 10.1371/journal.pone.0051185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Winchester L, Veeranki S, Givvimani S, Tyagi SC. Exercise mitigates the adverse effects of hyperhomocysteinemia on macrophages, MMP-9, skeletal muscle, and white adipocytes. Can J Physiol Pharmacol. 2014;92:575–582. doi: 10.1139/cjpp-2014-0059. [DOI] [PubMed] [Google Scholar]
  • 41.Winchester LJ, Veeranki S, Pushpakumar S, Tyagi SC. Exercise mitigates the effects of hyperhomocysteinemia on adverse muscle remodeling. Physiol Rep. 2018;6:e13637. doi: 10.14814/phy2.13637. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Chan SH, Hung CH, Shih JY, et al. Exercise intervention attenuates hyperhomocysteinemia-induced aortic endothelial oxidative injury by regulating SIRT1 through mitigating NADPH oxidase/LOX-1 signaling. Redox Biol. 2018;14:116–125. doi: 10.1016/j.redox.2017.08.016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Chacon D, Fiani B. A review of mechanisms on the beneficial effect of exercise on atherosclerosis. Cureus. 2020;12:e11641. doi: 10.7759/cureus.11641. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Aengevaeren VL, Mosterd A, Sharma S, et al. Exercise and coronary atherosclerosis: observations, explanations, relevance, and clinical management. Circulation. 2020;141:1338–1350. doi: 10.1161/CIRCULATIONAHA.119.044467. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Wang Y, Xu D. Effects of aerobic exercise on lipids and lipoproteins. Lipids Health Dis. 2017;16:132. doi: 10.1186/s12944-017-0515-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Li Y, Sun D, Zheng Y, Cheng Y. Swimming exercise activates aortic autophagy and limits atherosclerosis in ApoE-/- mice. Obes Res Clin Pract. 2020;14:264–270. doi: 10.1016/j.orcp.2020.04.008. [DOI] [PubMed] [Google Scholar]
  • 47.Xu Z, Zhang M, Li X, et al. Exercise ameliorates atherosclerosis via up-regulating serum β-hydroxybutyrate levels. Int J Mol Sci. 2022;23:3788. doi: 10.3390/ijms23073788. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Lai X, Zhang Y, Li M, et al. The effect of Shen-Yuan-Dan capsule on autophagy-related gene Atg13 promoter methylation and genomic methylation levels in atherosclerotic mice. Acta Cardiol Sin. 2020;36:260–267. doi: 10.6515/ACS.202005_36(3).20190906B. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Oppi S, Luscher TF, Stein S. Mouse models for atherosclerosis research-which is my line? Front Cardiovasc Med. 2019;6:46. doi: 10.3389/fcvm.2019.00046. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Dayal S, Lentz SR. Murine models of hyperhomocysteinemia and their vascular phenotypes. Arterioscler Thromb Vasc Biol. 2008;28:1596–1605. doi: 10.1161/ATVBAHA.108.166421. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Li Y, Zhang CG, Wang XH, Liu DH. Progression of atherosclerosis in ApoE-knockout mice fed on a high-fat diet. Eur Rev Med Pharmacol Sci. 2016;20:3863–3867. [PubMed] [Google Scholar]
  • 52.Thampi P, Stewart BW, Joseph L, et al. Dietary homocysteine promotes atherosclerosis in apoE-deficient mice by inducing scavenger receptors expression. Atherosclerosis. 2008;197:620–629. doi: 10.1016/j.atherosclerosis.2007.09.014. [DOI] [PubMed] [Google Scholar]
  • 53.Kapoor P, Ansari MN, Bhandari U. Modulatory effect of curcumin on methionine-induced hyperlipidemia and hyperhomocysteinemia in albino rats. Indian J Exp Biol. 2008;46:534–540. [PubMed] [Google Scholar]
  • 54.Obeid R, Herrmann W. Homocysteine and lipids: S-adenosyl methionine as a key intermediate. FEBS Lett. 2009;583:1215–1225. doi: 10.1016/j.febslet.2009.03.038. [DOI] [PubMed] [Google Scholar]
  • 55.Kolwicz SC, MacDonnell SM, Renna BF, et al. Left ventricular remodeling with exercise in hypertension. Am J Physiol Heart Circ Physiol. 2006;297:H1361–H1368. doi: 10.1152/ajpheart.01253.2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 56.Lee YI, Cho JY, Kim MH, et al. Effects of exercise training on pathological cardiac hypertrophy related gene expression and apoptosis. Eur J Appl Physiol. 2006;97:216–224. doi: 10.1007/s00421-006-0161-5. [DOI] [PubMed] [Google Scholar]
  • 57.Cao Z, Zhang Y, Sun T, et al. Homocysteine induces cardiac hypertrophy by up-regulating ATP7a expression. Int J Clin Exp Pathol. 2015;8:12829–12836. [PMC free article] [PubMed] [Google Scholar]
  • 58.Yu S, Chen Y, Yang H, et al. Hyperhomocysteinemia accompany with metabolic syndrome increase the risk of left ventricular hypertrophy in rural Chinese. BMC Cardiovasc Disord. 2020;20:44. doi: 10.1186/s12872-020-01350-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 59.Verboven M, Cuypers A, Deluyker D, et al. High intensity training improves cardiac function in healthy rats. Sci Rep. 2019;9:5612. doi: 10.1038/s41598-019-42023-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 60.Yan Z, Zeng N, Li J, et al. Cardiac effects of treadmill running at different intensities in a rat model. Front Physiol. 2021;12:774681. doi: 10.3389/fphys.2021.774681. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 61.Qipshidze N, Tyagi N, Sen U, et al. Folic acid mitigated cardiac dysfunction by normalizing the levels of tissue inhibitor of metalloproteinase and homocysteine-metabolizing enzymes postmyocardial infarction in mice. Am J Physiol Heart Circ Physiol. 2010;299:H1484–H1493. doi: 10.1152/ajpheart.00577.2010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 62.Li W, Tang R, Ouyang S, et al. Folic acid prevents cardiac dysfunction and reduces myocardial fibrosis in a mouse model of high-fat diet-induced obesity. Nutr Metab (Lond) 2017;14:68. doi: 10.1186/s12986-017-0224-0. [DOI] [PMC free article] [PubMed] [Google Scholar]

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