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. 2025 May-Jun;39(3):1262-1274.
doi: 10.21873/invivo.13930.

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

Yuko Onishi  1   2 , Naoki Miura  3 , Akihide Tanimoto  4 , Hiroaki Kawaguchi  5
Affiliations

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

Yuko Onishi et al. In Vivo. 2025 May-Jun.

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: Atherosclerosis; animal model; folic acid; high-fat and high-cholesterol diet; hyperhomocysteinemia; leucovorin; methotrexate; microminipig.

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Conflict of interest statement

The Authors declare that no conflicts of interest exist.

Figures

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.
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.
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.
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