ALYREF inhibits ferroptosis in vascular endothelial cells and improves atherosclerosis by epigenetic modification of CISD1
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Jinghai Hua1,2, Ling Yu1,2, Wenjun Xiong1,2
1Department of Cardiology, The First Affiliated Hospital, Jiangxi Medical College, Nanchang University, No. 17, Yongwai Zheng Street, Nanchang, 330006, Jiangxi, China.
Background:
Atherosclerosis (AS) is a disease marked by lipid metabolism dysfunction. Methylation of 5-methylcytosine (m5C) mRNA can regulate AS progression. We investigated the role and mechanism of m5C reader ALYREF in AS.
Methods:
ApoE-/- mice AS models were constructed. Oil Red O staining evaluated the degree of aortic plaque. Serum LDL and TG levels were assessed. ELISA detected vascular inflammation and ATP production. Expressions of ALYREF, CISD1, and ferroptosis-related proteins were detected by RT-qPCR and Western blot. CCK-8, EdU, and flow cytometry were used to detect cell viability and apoptosis. RIP assay validated the direct binding of ALYREF to CISD1. Mitochondrial morphology was observed by transmission electron microscopy (TEM). Mitochondrial membrane potential was determined by JC-1. Mitochondrial ROS and cytoplasmic ROS were tested by immunofluorescence staining. Oxidative stress damage (MDA), antioxidant enzymes (SOD/GSH), and Fe2+ levels were detected by kits. Methylated RNA was immunoprecipitated with m5C-specific antibody (MeRIP).
Results:
ALYREF expression declined in AS mice and human primary coronary artery endothelial cells (HCAEC) induced by oxidized low-density lipoprotein (ox-LDL). Elevated ALYREF improved ox-LDL-induced HCAEC apoptosis, inflammation, and lipid metabolism. ALYREF elevation attenuated mitochondrial damage and ferroptosis in ox-LDL-exposed HCAEC. ALYREF facilitated the stability and expression of CISD1 mRNA through m5C methylation. Reversing CISD1 expression negated the protective effects of ALYREF overexpression against ox-LDL-induced HCAEC damage. ALYREF-mediated epigenetic modification of CISD1 alleviated AS progression by reducing lipid levels and inhibiting ferroptosis in vivo.
Conclusion:
By enhancing m5C modification, ALYREF enhances the stability and expression of CISD1 mRNA, which impedes lipid metabolism and endothelial cell ferroptosis in AS and alleviates AS-associated pathological changes.
Insights
Methyl-N6-adenosine (m5C) reader ALYREF stabilizes CISD1 mRNA, reducing lipid accumulation and ferroptosis to alleviate atherosclerosis progression. This finding offers a novel therapeutic target for cardiovascular disease.
Area of Science:
- Biochemistry
- Molecular Biology
- Cardiovascular Research
Background:
- Atherosclerosis (AS) involves lipid metabolism dysfunction.
- Messenger RNA (mRNA) methylation, specifically 5-methylcytosine (m5C), plays a regulatory role in AS progression.
- The function of the m5C reader protein ALYREF in AS remains largely unexplored.
Purpose of the Study:
- To investigate the role and underlying mechanism of the m5C reader ALYREF in the development and progression of atherosclerosis.
- To determine how ALYREF influences lipid metabolism, inflammation, and cell death pathways in the context of AS.
Main Methods:
- Atherosclerosis models in ApoE-/- mice and human primary coronary artery endothelial cells (HCAEC) exposed to oxidized low-density lipoprotein (ox-LDL).
- Assessment of aortic plaque, serum lipid levels, vascular inflammation, and cellular ATP production.
- Analysis of ALYREF, CISD1, and ferroptosis-related protein expression via RT-qPCR and Western blot.
- Cellular assays for viability, apoptosis, mitochondrial function, reactive oxygen species (ROS), and oxidative stress markers.
- RNA immunoprecipitation (RIP) assay to confirm ALYREF binding to CISD1 mRNA and m5C RNA immunoprecipitation (MeRIP).
Main Results:
- ALYREF expression was significantly decreased in AS mouse models and ox-LDL-treated HCAEC.
- Overexpression of ALYREF ameliorated ox-LDL-induced apoptosis, inflammation, and lipid accumulation in HCAEC.
- ALYREF attenuated mitochondrial damage and ferroptosis in AS models.
- ALYREF directly enhanced the stability and expression of CISD1 mRNA through m5C modification.
- The protective effects of ALYREF were dependent on CISD1 expression, as its reversal negated ALYREF's benefits.
Conclusions:
- ALYREF plays a protective role in atherosclerosis by enhancing m5C modification of CISD1 mRNA, thereby increasing its stability and expression.
- This ALYREF-mediated mechanism impedes aberrant lipid metabolism and endothelial cell ferroptosis, alleviating AS-associated pathological changes.
- Targeting ALYREF-CISD1 interaction presents a potential therapeutic strategy for managing atherosclerosis.
