Research ArticleExperimental Studies
Open Access

Methotrexate Enhances Atherosclerosis Progression via Impairment of Folate Pathway in a Microminipig Model

YUKO ONISHI, NAOKI MIURA, AKIHIDE TANIMOTO and HIROAKI KAWAGUCHI
In Vivo May 2025, 39 (3) 1262-1274; DOI: https://doi.org/10.21873/invivo.13930

Abstract

Background/Aim: As the pathophysiology of Microminipigs (μMPs) is similar to that of human, μMPs are useful in atherosclerosis research. To clarify the effect of methotrexate (MTX) on atherosclerosis, we investigated the pathology of MTX-induced atherosclerosis lesion exacerbation in μMPs fed a high-fat and high-cholesterol diet (HFHCD).

Materials and Methods: The μMPs were divided into four groups: HFHCD, HFHCD+MTX, HFHCD+MTX+leucovorin (LV), and HFHCD+MTX+folic acid (FA), and fed for two weeks. Laboratory tests including blood lipid, FA, and homocysteine (Hcy) levels, and pathological evaluation of the atherosclerosis lesion area and thickness were performed. Hepatic and jejunal gene expressions related to lipid and folate metabolism pathways including 5-methyltetrahydrofolate-homocysteine methyltransferase (MTR) were monitored using RT-PCR.

Results: The HFHCD+MTX group showed increased blood Hcy (p<0.01) and decreased FA levels (p<0.05) in accordance with increased hepatic MTR mRNA expression (p<0.1) and exacerbation of atherosclerosis (p=0.051 for lesion area and p=0.045 for lesion thickness) compared to the HFHCD group. Administration of LV or FA attenuated the MTX-induced increase in the Hcy level (p<0.01), atherosclerosis lesion thickness (p<0.1), and MTR mRNA expression (p<0.1 in HFHCD+MTX vs. HFHCD+MTX+LV groups).

Conclusion: MTX exacerbated HFHCD-induced atherosclerosis mediated through reduced blood FA and the subsequent increase of Hcy in μMPs, indicating that the μMP model may advance cardio-oncology research by providing useful experimental approaches. As MTX is administered for rheumatoid arthritis and malignant tumors in humans, atherosclerosis exacerbation should be acknowledged as a possible adverse effect of MTX treatment.

Keywords:

Introduction

Atherosclerosis is a predominant risk factor in cardiovascular and cerebrovascular events and closely related to serious morbidity (1). Myocardial and cerebral infarction due to arterial atherosclerosis are major causes of death, and result in a discrepancy between average and healthy life expectancy (2). Folic acid (FA) deficiency causes blood homocysteine (Hcy) elevation, a risk factor for atherosclerotic vascular diseases in humans (3, 4). Therefore, blood Hcy levels can be used as prognostic indicators along with non-high-density lipoprotein (HDL)-cholesterol levels (5, 6). Furthermore, blood Hcy levels are high in countries with low folic acid intake (7). Epidemiological studies also demonstrated that increased blood Hcy (hyperhomocysteinemia) is a risk factor for human infarct diseases (8-11). Methotrexate (MTX), a widely used drug for cancer and rheumatoid arthritis (RA) patients, is an FA metabolism antagonist that reduces blood FA levels (12, 13), suggesting that MTX could exacerbate atherosclerosis. However, conversely, MTX reportedly protects against atherosclerosis and cardiovascular events in RA patients (14, 15). In addition to clinical cohort studies, experimental approaches using appropriate animal atherosclerosis models are required to resolve whether MTX is actually atheroprotective or proatherogenic.

To investigate the pathogenesis of atherosclerosis, appropriate animal models that reproduce human physiology and pathology are required. Mice and rabbits were widely used to develop animal models of atherosclerosis, but lipid metabolism in mice is high-density lipoprotein (HDL)-cholesterol dominant and typically resistant to a high-fat/high-cholesterol diet (HFHCD). Therefore, gene knockout is necessary to cause atherosclerosis in mice (16, 17). Rabbits are more sensitive to HFHCD for the introduction of atherosclerosis, and have a similar lipid metabolism to humans. The use of low-density lipoprotein receptor (LDLr) gene-mutated Watanabe heritable hypercholesterolemia and transgenic rabbits clarified the effects of specific genes on the development of atherosclerosis (18-20). However, swine provide useful animal models, because their physiology and style of feeding and sleep are similar to those of humans, leading to the development of many swine strains for experimental uses, including atherosclerosis research (21). Among them, the Microminipig™ (μMP, Fuji Micra, Shizuoka, Japan) has been established as the world’s smallest experimental minipig (22). Our previous studies demonstrated that μMPs are very sensitive to HFHCD and prone to develop hypercholesterolemia-induced atherosclerosis (23-25).

In the present study, we investigated the potential for MTX to exacerbate atherosclerosis in a 2-week μMP hyperlipidemia-induced atherosclerosis model. As FA are administered to prevent FA antagonistic toxicity of MTX in RA patients (26), leucovorin (LV) and FA are used to realize a reduction of MTX-induced blood Hcy elevation and atherosclerotic lesions. Herein, we demonstrated that MTX accelerated atherosclerosis via decreased FA and a subsequent increase of blood Hcy in μMP models. These results indicate that short-term MTX administration would have the adverse effect of atherosclerosis lesion exacerbation. The present study using the μMP atherosclerosis model would promote cardio-oncology research by providing an essential tool for studying atherosclerosis associated with anticancer treatment.

Materials and Methods

Animal maintenance. Male μMPs were obtained from a breeder (Fuji Micra) and maintained in animal rooms at a temperature of 20-26°C and relative humidity of 30-70%, with a 12-h light/dark cycle. Tap water was available ad libitum. All experimental protocols were approved by the Ethics Committee of Animal Care and Experimentation, Kitasato University (21-016) and HAMRI Co., Ltd. (IB20032). The research was performed in accordance with the Institutional Guidelines for Animal Experiments and in compliance with the Japanese Law Concerning the Protection and Control of Animals (Law No. 105 and Notification No. 6).

Study design. Twelve μMPs (6-7 months old, 10.6±0.85 kg BW) were divided equally into four groups and each fed HFHCD for 2 weeks, involving 300 g diet a day ingested in the morning. The HFHCD diet was composed of fat (3.6 g/kg/day, refined lard; Miyoshi Oil & Fat Co., Tokyo, Japan) and cholesterol (0.15 g/kg/day, Wako Pure Chemical Industries, Osaka, Japan) mixed with a regular diet (Marubeni Nisshin Feed, Tokyo, Japan), as previously reported (23). In HFHCD+MTX, HFHCD+MTX+LV, and HFHCD+MTX+FA groups, MTX (3.3 mg/body/day), LV (0.99 mg/body/day), and FA (4.5 mg/body/day) were administered intramuscularly in the morning, respectively (Table I). LV, also known as folic acid, is an active form of folic acid that does not require activation by dihydrofolate reductase.

View this table:
Table I.

Body weight and visceral and subcutaneous adiposity in the μMP model.

Laboratory tests. Clinical observation was conducted every day, and body weight was measured on the first and 14th days of the experiment. Peripheral blood samples were collected from the cranial vena cava at Days 1 and 14 for hematology and biochemistry. Biochemical parameters included aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, lactate dehydrogenase, γ-glutamyl transpeptidase, total bilirubin, and glucose. Levels of total cholesterol (TC), very low-density lipoprotein-cholesterol (VLDL-C), low-density lipoprotein-cholesterol cholesterol (LDL-C), high-density lipoprotein-cholesterol (HDL-C), and triglycerides (TG) were analyzed using an automated apparatus (LABOSPECT 008, Hitachi High-Tech Corporation, Tokyo, Japan; DM-JACK, Minaris Medical Co., Ltd., Tokyo, Japan). After the 2-week experiment, all μMPs were anesthetized, sacrificed by bilateral axillary artery exsanguination, and necropsied. The weight of visceral fat (greater omentum and mesenterium) was measured and calculated as the relative adiposity weight [fat weight (g)/BW (kg)].

Pathological examination. At necropsy, the aorta, heart, liver, kidneys, spleen, and intestine were resected for pathological examination. The heart, liver, kidneys, spleen, and visceral adipose tissue (greater omental and mesenteric adipose tissues) were weighed. All organs were fixed in 10% phosphate-buffered formalin and embedded in paraffin for routine histological examination. The thoracic and abdominal aortas were cut open longitudinally and stained with Oil Red O stain. En face images of the aortas were captured using a digital camera, and the Oil Red O-positive area relative to the whole surface area was calculated using Image J software (23, 24). Formalin-fixed, paraffin-embedded aortas were processed for hematoxylin and eosin (H&E) and Elastica Masson stains. The abdominal aorta was traversed at 3-mm intervals, and 17-24 sections/animal were subjected to pathological examination. Then, three of the most severe lesion sites in the abdominal aorta were arbitrarily selected, and the thickness of each lesion site was measured at three locations, wherein the lesion thickness was averaged to give a value of one individual. Immunostaining for abdominal atherosclerotic lesions was carried out for paraffin sections using antibodies against smooth muscle actin (anti-α-SMA clone 1A4, ×50; Dako, Tokyo, Japan) and macrophages (anti-lysozyme rabbit polyclonal antibody, ×25; Dako, Tokyo, Japan).

Quantitative reverse transcription polymerase chain reaction (qRT-PCR). The liver and jejunum were collected immediately following necropsy in all animals (n=3/group). Small pieces of fresh tissues (~3 mm3) were placed in RNAlater (Sigma-Aldrich, Tokyo, Japan) at 4°C overnight, and then stored at −30°C until use. Total RNA was extracted from the liver and jejunum using RNeasy Mini Kit (Qiagen, Hilden, Germany), and mRNA expression of LDLr, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), sterol regulatory element binding protein 2 (SREBP2), Niemann-Pick C1-Like 1 (NPC1L1), 5-methyltetra-hydrofolate-homosystein methyltransferase (MTR), choline dehydrogenase (CHDH), and phosphatidylethanolamine N-methyltransferase (PEMT) in the liver was quantified using qRT-PCR. Expression of jejunal apolipoprotein B-100 RNA editing catalytic subunit-1 (APOBEC-1) mRNA was also quantified using qRT-PCR (23, 24). The expression level of glyceraldehyde-3-phosphate dehydrogenase was used as an internal control. Primers used for RT-PCR are listed in Table II.

View this table:
Table II.

Genes investigated and primers used for PCR.

Statistics. All results are expressed as the mean±standard error. Differences were analyzed using t-tests. Differences were considered significant at p<0.05 and borderline-significant at p>0.05 and <0.1.

Results

General observation. No abnormal manifestations were observed in any of the groups during the experimental period. The final BW gains (from Day 1 to Day 14, with starting experimental value of 100%) in all groups were approximately 10% (Table I). BW gains and relative adiposity weights showed no significant differences among the four groups, except for between HFHCD+MTX and HFHCD+MTX+LV groups, with p=0.092 and p=0.065, respectively (Table I). There were no significant differences in mean absolute or relative organ weights.

Laboratory tests. Blood levels of TC, LDL-C, HDL-C, and free-C increased rapidly during the 2 weeks in all groups. TC, LDL-C, and free-C levels increased markedly in the HFHCD group than in the other groups; however, no significant statistical differences were observed (Figure 1). Blood levels of hepatic enzymes showed no significant differences among the four groups, and were within the reference data on μMPs (27, 28). The blood FA concentration in the HFHCD+MTX group was lower than that in the HFHCD group (p=0.025), while the concentrations in HFHCD+ MTX+LV and HFHCD+MTX+FA groups were comparable with those in the HFHCD group (Figure 2A). The blood Hcy level in the HFHCD+MTX group was significantly higher than in all other groups (p<0.01). Hcy levels in HFHCD+MTX+LV and HFHCD+MTX+FA groups were moderately decreased compared with the HFHCD group (p <0.05) (Figure 2B). These results indicate that additional administration of LV and FA attenuated the MTX-induced blood Hcy elevation.

Figure 1.
Figure 1.

Serum levels of lipid metabolism markers. (A) TC, (B) LDL-C, (C) HDL-C, (D) Free-C. Blood levels of all markers increased rapidly during the 2 weeks in all groups. Open circle: HFHCD, closed circle: HFHCD+MTX, open rectangle: HFHCD+MTX+LV, closed rectangle: HFHCD+MTX+FA. TC: Total cholesterol; LDL-C: low-density lipoprotein-cholesterol, HDL-C: high-density lipoprotein-cholesterol, Free-C: free cholesterol.

Figure 2.
Figure 2.

Serum folic acid and homocysteine levels. (A) FA and (B) Hcy concentration at 2 weeks. In the HFHCD+MTX group, FA concentration decreased, and Hcy concentration increased compared to other groups. *p<0.05, **p<0.01. FA: Folic acid; Hcy: homocysteine; HFHCD: high-fat and high-cholesterol diet; MTX: methotrexate; LV: leucovorin.

Pathological examination. None of the animals showed grossly significant anomalies in any of the organs, except for the aortas and arteries. En face Oil red O staining of the thoracic and abdominal aortas stained linear and patchy atherosclerotic lesions red in color (Figure 3A). The lesion % area with positive Oil red O staining in the HFHCD+MTX group was larger than the HFHCD group (p=0.051, Figure 3B). The HFHCD+MTX group showed no significant differences in the lesion % area between HFHCD+MTX+LV and HFHCD+MTX+FA groups. In contrast, the thickness of atherosclerotic lesions in the aortas of the HFHCD+MTX group, which represent the most severe atherosclerotic lesions, were thicker than in the HFHCD group (p=0.045) (Figure 3C). The lesion thickness in the HFHCD+MTX+LV and HFHCD+MTX+FA groups were thinner than in the HFHCD+MTX group, with p=0.074 and p=0.082, respectively (Figure 3C). The atherosclerotic lesions were localized in the arterial and aortic intima, which was thickened due to proliferation of lysozyme-positive macrophage-derived foam cells and α-SMA-positive smooth muscle cells (Figure 4A, B, and C). No lymphoproliferative diseases were observed in MTX-treated groups.

Figure 3.
Figure 3.

Atheromatous lesions of aorta. (A) Oil red O-stained atheromatous lesions: Circles are intensely red atheromatous lesions. (B) En face analysis of Oil red O-stained atheromatous lesions: The lesions were increased in HFHCD+MTX, HFHCD+MTX+FA groups compared with the HFHCD group. (C) Thickness of the atheromatous lesion in the abdominal aorta: Plaque thickness was greatest in the HFHCD+MTX group compared with the other groups. *p<0.05, #p<0.1. HFHCD: High-fat and high-cholesterol diet; MTX: methotrexate; LV: leucovorin; FA: folic acid.

Figure 4.
Figure 4.

Histopathological and immunohistochemical examinations of atherosclerotic lesions in HFHCD+MTX group. (A) Right coronary artery: Atheromatous plaque is shown. Arrow indicates internal elastic lamina. (B) Left coronary artery: Intimal infiltration of foam cells (arrowhead) is shown. (C, D) The abdominal aorta showed intimal fibrosis. (E) The lesion of intimal fibrosis included a few lysozyme-positive cells (arrowheads). (F) Some intima cells in the abdominal aorta were positive for α-SMA. A, B, and C: H&E stain, D: Elastica-Masson stain, E and F: Immunohistochemical stain. HFHCD: High-fat and high-cholesterol diet; MTX: methotrexate; H&E: hematoxylin and eosin.

Study of gene expression. The expression level of hepatic MTR mRNA in the HFHCD+MTX group was higher than in the HFHCD group (p=0.085). The MTR mRNA expression level in the HFHCD+MTX+LV group was decreased in comparison with that in the HFHCD+MTX group (p=0.099), and similar to that in the HFHCD group. The MTR mRNA expression level in the HFHCD+MTX+FA group was comparable with that in the HFHCD group (Figure 5A). Expression levels of hepatic CHDH mRNA in the HFHCD group showed differences among HFHCD+MTX+LV (p=0.02), HFHCD+MTX (p=0.066), and HFHCD+MTX+FA groups (p=0.099) (Figure 5B). PEMT showed no significant differences among the four groups (Figure 5C). Expression of hepatic LDLr mRNA was high in the HFHCD group, but this was not statistically significant. A greater increase in LDLr mRNA expression was observed in the HFHCD+MTX+FA group compared to the HFHCD group (p=0.028) (Figure 6A). The expression of hepatic NPC1L1 mRNA was higher in the HFHCD+ MTX+FA group than HFHCD group (p=0.098) (Figure 6B). Hepatic HMGCR and intestinal APOBEC-1 mRNA expression levels showed no differences among the four groups (Figure 6C and D).

Figure 5.
Figure 5.

Expression of hepatic MTR, CHDH, and PEMT mRNA. (A) MTR was up-regulated in the HFHCD+MTX group and down-regulated in HFHCD+MTX+LV and HFHCD+MTX+FA groups. (B) CHDH was up-regulated in HFHCD+MTX and HFHCD+MTX+LV groups and down-regulated in the HFHCD+MTX+FA group. (C) PMET: No difference was found among the 4 groups. *p<0.05, #p<0.1. MTR: 5-Methyltetrahydrofolate-homosystein methyltransferase; CHDH: choline dehydrogenase; PEMT: phosphatidylethanolamine N-methyltransferase; HFHCD: high-fat and high-cholesterol diet; MTX: methotrexate; LV: leucovorin; FA: folic acid.

Figure 6.
Figure 6.

Expression of hepatic LDLr, HMGCR, NPC1L1, and jejunal APOBEC-1 mRNA. (A) Increased LDLr mRNA expression was observed in the HFHCD+MTX+FA group compared with the HFHCD group. (B) The expression of NPC1L1 was higher in the HFHCD+MTX+FA group than in the HFHCD group. Expressions of hepatic LDLr (C) and jejunal APOBEC-1 (D): No difference was found among the 4 groups. *p<0.05, #p<0.1. HFHCD: High-fat and high-cholesterol diet; MTX: methotrexate; LV: leucovorin; FA: folic acid; LDLr: low-density lipoprotein receptor; HMGCR: 3-hydroxy-3-methylglutaryl-coenzyme A reductase; NPC1L1: Niemann-Pick C1-Like 1; APOBEC-1: apolipoprotein B-100 RNA editing catalytic subunit-1.

Discussion

Herein, we clearly reproduced our previous studies in which hypercholesterolemia-induced atherosclerosis developed in μMPs within 2 weeks of feeding them HFHCD (23-25). This is a much shorter experimental period to produce macroscopically identifiable atherosclerotic lesions than in other pig atherosclerosis models (29-32). To the best of our knowledge, this study is the first to demonstrate MTX-induced atherosclerosis exacerbation via impairment of folic acid metabolism in a hyperlipi-demic animal model.

HFHCD feeding increased TC, LDL-C, and free-C levels approximately 5-fold or more, as previously reported (23). MTX administration, however, led to no additional increase in blood lipid levels. However, MTX treatment decreased blood FA and increased Hcy with a subsequent increase of atherosclerosis lesions. Moreover, LV and FA treatments attenuated blood Hcy levels and atherosclerosis lesion thickness. These results indicate that MTX accelerates arteriosclerosis via increased blood Hcy (hyperhomocysteinemia). LV or FA supplementation as MTX antagonists administered to HFHCD+MTX-fed μMPs decreased atherosclerosis lesion thickness, but not lesion area, suggesting that LV or FA can suppress lesion progression of pre-existing atherosclerosis. As Hcy is a risk factor for worsening atherosclerosis in humans (8-11, 33), MTX medication is suggested to be a risk factor of atherosclerosis progression. The most important risk factor affecting atherosclerosis is blood LDL-C levels, which are mainly regulated by LDL-C removal via hepatic LDLr and cholesterol synthesis via hepatic HMGCR activity (34, 35). In the present μMP atherosclerosis model, MTX treatment did not change the LDLr, HMGCR mRNA, or blood lipid levels. MTX would exhibit no direct effects on lipid metabolism in μMPs; therefore, MTX-induced atherosclerosis progression would be mediated through hyperlipidemia-independent mechanism(s) in the present μMP model.

Some reports showed that treatment with FA and vitamin B12 is not associated with reduction of cardiovascular events (36, 37). In contrast, a meta-analysis demonstrated evidence for the beneficial effect of FA and vitamin B supplementations in reducing Hcy and preventing the combined risk of stroke, myocardial infarction, and vascular death (38). Although the preventive effects of FA on human atherosclerosis remain controversial, FA supplementation was found to reduce atherosclerotic lesion thickness in accordance with blood Hcy reduction in the μMP model. The expression of hepatic MTR, an enzyme that catalyzes the conversion of Hcy to methionine, was up-regulated in MTX-treated μMPs and down-regulated by LV and FA. This enzymatic activity requires 5-methyltetrahydrofolate (5-MTHF), a metabolite produced in the folate metabolic pathway. MTX primarily targets dihydrofolate reductase, reduces blood FA, and subsequently suppresses 5-MTHF production. Then, decreased 5-MTHF leads to increased Hcy via decreased MTR enzymatic activity. In the present study, MTR mRNA expression was affected by MTX, suggesting that the folate metabolic pathway would be comparable between humans and μMPs.

Clinically, MTX is a first-line treatment for RA patients (39). As the present study indicated, MTX increases blood Hcy levels via reduced blood FA (13, 40); therefore, MTX is suggested to be a risk factor for atherosclerosis and related cardiovascular events. For RA patients receiving MTX treatment, FA is administered as a supplement and this decreases Hcy levels and protects against cardiovascular events (15, 40). On the contrary, there are multimodal effects of MTX on anti-inflammation, decrease in proatherogenic lipoprotein(a), and improvement of endothelial functions that prevent atherosclerosis progression (41-44). Actually, MTX treatment is associated with a decreased cardio-vascular risk in RA patients (26, 44, 45). Conversely, the prescription of immunomodulatory drugs including MTX results in an increased risk of cardiovascular events of myocardial infarction, stroke, and cardiovascular death in patients with peripheral artery diseases (46). We cannot directly extrapolate the results obtained from our μMP model to human atherosclerosis pathology to conclude whether MTX is atheroprotective or proatherogenic. However, as one possible explanation, the discrepancy between clinical and experimental observations may be attributable to differences in the dose and duration of MTX administration. In the present μMP model, the MTX dose and duration (3.3 mg/body/day for 2 weeks) were much lower and shorter, respectively, than in the standard protocol for RA patients (47). Another possibility is that the presence or absence of complications of other diseases like RA might affect the effect of MTX on atherogenesis. As RA and atherosclerosis share the same pathophysiological features as chronic inflammatory diseases and exacerbation or progression of atherosclerosis may occur in RA patients (48), further clinical and experimental evaluations would be necessary to clarify the effects of MTX on atherosclerosis.

In conclusion, MTX exacerbates hyperlipidemia-induced atherosclerosis via impairment of the folate metabolic pathway but independently by enhancement of hyperlipidemia in the μMP model. This animal model is useful for pathological studies of atherosclerosis associated with MTX treatment as well as pharmacological evaluation of HMGCR inhibitors (49). Cardio-oncology is an emerging interdisciplinary field dedicated to the early detection and treatment of adverse cardiovascular events associated with anticancer treatment (50). The present study may promote cardio-oncology research by providing a suitable animal model.

Acknowledgements

This research was partly supported by the Japan Society for the Promotion of Science (JSPS) Kakenhi (grant numbers: 16K08023 and 20K06395). We are grateful to Mr. H. Tashiro, Ms. T. Wada, and Mr. R. Sato (Kitasato University), as well as Dr. H. Kawasaki (Astellas Pharm, Inc.) for their valuable assistance.

Footnotes

  • Authors’ Contributions

    Designed the study: YO, NM, AT, and HK. Performed research: YO and HK. Collected and analyzed data: YO and HK. Wrote the paper: YO, NM, AT, and HK.

  • Conflicts of Interest

    The Authors declare that no conflicts of interest exist.

  • Received January 19, 2025.
  • Revision received February 5, 2025.
  • Accepted February 11, 2025.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).

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Methotrexate Enhances Atherosclerosis Progression via Impairment of Folate Pathway in a Microminipig Model
YUKO ONISHI, NAOKI MIURA, AKIHIDE TANIMOTO, HIROAKI KAWAGUCHI
In Vivo May 2025, 39 (3) 1262-1274; DOI: 10.21873/invivo.13930
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