Vitamin D supplementation inhibits atherosclerosis through repressing macrophage-induced inflammation via SIRT1/mTORC2 signaling

To the Editor: Peripheral artery disease (PAD) is a prevalent vascular condition characterized by arterial narrowing or occlusion, primarily due to atherosclerosis. Approximately 17.5% of patients with PAD develop acute lower limb ischemia due to vulnerable atherosclerotic plaque rupture, which significantly impacts their quality of life.[1] A key feature of vulnerable plaque is pronounced macrophage infiltration into the fibrous cap, which drives a robust inflammatory response.[2] Previous studies have demonstrated that vitamin D receptor (VDR) exerts atheroprotective effects by modulating macrophage functions.[3,4] However, the role of VDR in macrophage inflammatory responses and atherosclerosis remains elusive. In this study, we demonstrated that VDR activates silent information regulator transcript-1 (SIRT1) transcription and subsequently reduces interleukin-1 beta (IL-1β) expression in macrophages. Besides, vitamin D supplementation inhibited the progression of atherosclerosis and enhanced the stability of atherosclerotic plaques in a mouse model. Materials and methods are described in detail in the Supplementary Materials, https://links.lww.com/CM9/C513. All specimens were obtained from the Biobank of Renji Hospital, School of Medicine, Shanghai Jiaotong University. The study was conducted in accordance with the approval of the research Ethics Committee of Renji Hospital (No. KY2021-129-B), and written informed consent was obtained from all patients. The animal protocols were approved by the Committee on the Ethics of Animal Experiments at the School of Medicine, Shanghai Jiaotong University (No. RA-2020-372). To identify the genes potentially associated with atheroslcerosis and plaque vulnerability, we analyzed the transcriptome of macrophages from human vulnerable and stable atherosclerotic plaque samples. Gene Set Enrichment Analysis (GSEA) results showed that vitamin D-associated genes were significantly enriched in vulnerable plaque [Supplementary Figure 1, https://links.lww.com/CM9/C513]. Furthermore, we found that the concentration of 25-hydroxyvitamin D3 in the serum of PAD patients was significantly lower compared to healthy donors, and we observed a gradual decline in the concentration of serum 25-hydroxyvitamin D3 in patients as the Rutherford classification increased. We also found that PAD patients with lower concentrations of serum 25-hydroxyvitamin D3 (≤20 ng/mL) had worse primary patency after endovascular treatment than those with higher concentrations of serum 25-hydroxyvitamin D3 (>20 ng/mL) [Supplementary Figure 2 and Supplementary Table 1, https://links.lww.com/CM9/C513]. To assess the impact of vitamin D deficiency on plaque progression and stability, we established a cohort of mice supplemented with a low-vitamin D diet. The results showed that the vitamin D deficiency led to a marked increase in mice’s plaque complexity, vulnerability, and macrophage apoptosis within the atherosclerotic plaques despite no significant changes in lipid metabolism or glucose regulation [Figure 1A–E, Supplementary Figure 3, https://links.lww.com/CM9/C513]. To further elucidate the mechanism through which vitamin D deficiency aggravates atherosclerosis, we found that vitamin D deficiency promotes macrophage apoptosis and the activation of pro-inflammatory pathways, specifically through increased IL-1β expression. In both clinical samples and mouse models, lower serum vitamin D3 levels were associated with elevated IL-1β in macrophages, and vitamin D supplementation effectively suppressed IL-1β expression [Supplementary Figure 4, https://links.lww.com/CM9/C513]. In vitro studies confirmed that vitamin D reduces IL-1β transcription and secretion in macrophages stimulated by lipopolysaccharide (LPS) or cholesterol crystals [Figure 1F, G, Supplementary Figure 5, https://links.lww.com/CM9/C513].Figure 1: Vitamin D supplementation inhibits atherosclerosis through repressing macrophage-induced inflammation via SIRT1/mTORC2 signaling. (A) Representative En face Oil Red O staining of aortas and quantification of plaque area in mice with or without vitamin D deficiency (n = 12 per group). (B–E) Cross-sections of aortic roots stained with Oil Red O and quantification of plaque area in mice with or without vitamin D deficiency (n = 12 per group) (B). Scale bar = 200 μm. Histological analysis of aortic root sections to quantify NC (C), vulnerability index (D), and apoptosis (TUNEL) in mice with or without vitamin D deficiency (n = 12 per group). Representative images are shown (E). Scale bar = 20 μm. (F, G) Macrophages were stimulated with 100 ng/mL LPS with or without 10−8 mmol/L vitamin D for 24 h. (F) Immunofluorescence staining of CD68 and IL-1β. (G) Intracellular pro-IL-1β and IL-1β levels were measured by immunoblotting. (H) Macrophages were stimulated with the mTORC2 inhibitor or control, and IL-1β expression was detected by immunofluorescence staining of CD68 and IL-1β. (I, J) Macrophages were stimulated with vitamin D (10−8 mmol/L) for 24 h, and the expression levels of Rictor and its mRNA were measured by immunoblotting and RT-qPCR, respectively. (K, L) Macrophages were stimulated with vitamin D (10−8 mmol/L) for 24 h, and the expression level of SIRT1 was measured by immunoblotting and immunofluorescence staining. (M) Macrophages were used to perform ChIP assays with the VDR antibody. Eluted DNA fragments were used as templates for RT-qPCR to measure the enrichment of VDR to the SIRT1 promoter. Signals were calculated as a percentage of input. (N) Macrophages were transfected with the SIRT1 plasmid, and the expression level of Rictor was measured by immunoblotting. (O) Macrophages were used to perform ChIP assays with the SIRT1 antibody. Eluted DNA fragments were used as templates for RT-qPCR to measure the enrichment of SIRT1 to the Rictor promoter. Signals were calculated as a percentage of input. (P) Macrophages transfected with SIRT1 plasmid were used to perform immunoprecipitation assays. The associated proteins were detected by NRF1 antibodies. (Q, R) Representative En face Oil Red O staining of aortas and the quantitative analysis of the plaque vulnerability index in mice with or without vitamin D supplementation (n = 12 per group). Plaque vulnerability index was measured in the mouse models of atherosclerotic plaque. Plaque composition, including calcification, was quantified by histological analysis of aortic root sections. Aortic root sections were stained with H&E, Masson staining, anti-CD68, and anti-α-SMA to assess plaque composition and vulnerability index. Scale bars = 100 μm. α-SMA: Alpha-smooth muscle actin; Ctrl: Control; ChIP: Chromatin immunoprecipitation; CD68: Cluster of differentiation 68; IL-1β: Interleukin-1 beta; LPS: Lipopolysaccharide; mTORC2: Mechanistic target of rapamycin complex 2; NC: Necrotic core; NRF1: Nuclear respiratory factor 1; RT-qPCR: Reverse transcription quantitative polymerase chain reaction; SIRT1: Sirtuin 1; VDR: Vitamin D Receptor; VitD: Vitamin D.Further mechanistic studies revealed that vitamin D exerts its anti-inflammatory effects through the mechanistic target of rapamycin complex 2 (mTORC2)/Ak strain transforming (Akt) /forkhead box O1 (FOXO1) axis. First, we demonstrated that mTORC2 inhibition reverses the protective role of vitamin D, leading to increased IL-1β production. To further confirm the association between vitamin D and mTORC2, we stimulated the macrophages with vitamin D, and results showed elevated mTORC2 expression in macrophages [Figure 1H–J]. Besides, we found that vitamin D supplementation significantly induced Akt activation and FOXO1 phosphorylation. Consequently, an increased presence of cytoplasmic FOXO1 was noted in vitamin D-stimulated macrophages. These findings suggest that vitamin D exerts its inhibitory effect on IL-1β expression through the mTORC2/Akt/FOXO1 axis [Supplementary Figure 6, https://links.lww.com/CM9/C513]. To elucidate the mechanism by which vitamin D induces mTORC2 expression, we compared downregulated genes in PAD lesions with upregulated genes in vitamin D-stimulated macrophages and predicted Rictor promoter targets using the JASPAR database, https://jaspar.elixir.no/ and UCSC genome browser, https://jaspar.elixir.no/. Bioinformatics analyses identified 27 common potential genes. A green fluorescent protein (GFP)-IL-1β reporter system revealed that SIRT1 had the highest inhibitory effect on the IL-1β-GFP reporter [Supplementary Figure 7, https://links.lww.com/CM9/C513]. Vitamin D treatment increased SIRT1 expression and promoted its translocation to the nucleus. Chromatin immunoprecipitation (ChIP) assays confirmed VDR binding to the SIRT1 promoter, and SIRT1 overexpression enhanced Rictor levels. SIRT1 was found at the Rictor promoter, and immunoprecipitation assays showed SIRT1 interacts with NRF1. Together, these findings suggest that vitamin D enhances mTORC2 expression via the SIRT1/NRF1 axis [Figure 1K–P]. Finally, we assessed the impact of vitamin D supplementation on atherosclerosis in mice fed an high fat diet (HFD) for 8 weeks. Vitamin D treatment significantly reduced atherosclerotic lesion area and plaque vulnerability without affecting arterial calcification, glucose metabolism, serum lipids, body weight, or insulin resistance [Figure 1Q, R, and Supplementary Figure 8, https://links.lww.com/CM9/C513]. This study reveals that vitamin D deficiency promotes atherogenesis by inducing IL-1β-mediated vascular inflammation in mice. We provide the evidence that macrophage mTORC2 function is regulated by vitamin D-VDR signaling, with vitamin D deficiency suppressing VDR-mediated SIRT1 transcription and downregulating Rictor, leading to increased IL-1β production and vascular inflammation. Vitamin D supplementation reversed these effects by activating mTORC2 signaling. These findings highlight a novel mechanism through which vitamin D regulates macrophage inflammation and inhibits atherosclerosis progression. Our data show that vitamin D deficiency accelerates atherosclerotic lesion development and increases plaque vulnerability, despite similar lipid levels. Vitamin D did not affect body weight or vascular calcification, suggesting that its atheroprotective effects are independent of lipid or calcium metabolism. Elevated IL-1β production in vitamin D-deficient macrophages and the reduction of IL-1β by vitamin D treatment further emphasize the role of macrophage-driven inflammation in atherosclerosis progression. Previous studies showed an inverse relationship between vitamin D and vulnerable plaque markers such as metalloproteinases and leptin. Our findings demonstrate that vitamin D supplementation not only reduces atherosclerotic plaque formation but also enhances plaque stability by preventing pro-inflammatory cytokine release and reducing macrophage apoptosis. This suggests a novel anti-atherosclerotic role for vitamin D, with potential clinical implications in cardiovascular health. In summary, we provide evidence supporting vitamin D’s anti-atherosclerotic effects, partly through mTORC2 signaling activation in macrophages. Our findings pave the way for further research into the therapeutic potential of vitamin D and its analogs in preventing atherosclerosis. Funding This work was supported by grants from the National Nature Science Foundation of China (Nos. 82170511 and 82000447), the Nature Science Foundation of Shanghai City (No. 20ZR1471800), the China Postdoctoral Science Foundation (No. 2022M712111), the Cultivation Foundation of Renji Hospital (No. RJTJ24-QN-044), and the Project of Zhejiang Provincial Health Commission (No. 2024KY1621).