Hyperhomocysteinemia accelerates atherosclerosis through a B cell–derived anti-β2 glycoprotein I–mediated endothelial injury
H Chen, J Jin, Z Liang, H Lv, F Fan, Y ZhangAbstract
Background
Hyperhomocysteinemia (HHcy) accelerates atherosclerosis through immunometabolic and inflammatory mechanisms. Previous studies have shown that HHcy promotes the production of B cell–derived anti-β2 glycoprotein I (β2GPI) antibodies, which enhance macrophage inflammatory activation. However, whether HHcy-induced anti-β2GPI antibodies contribute to arterial endothelial injury and subsequent atherosclerotic progression remains unclear.
Methods
Four groups of ApoE⁻/⁻ mice were fed a high-fat diet for 4 weeks to establish atherosclerotic conditions. Mice were subsequently treated with homocysteine (Hcy; 1.8 g/L in drinking water) or rituximab (75 μg per 20 g body weight, intraperitoneally every other day) while continuing the high-fat diet for an additional 4 weeks. At week 8, plasma samples were collected to measure Hcy, cholesterol, anti-β2GPI IgG, and total IgG levels. The aorta and heart were harvested for Oil Red O staining and immunofluorescence analysis of VCAM-1, β2GPI, and CD31 to assess vascular inflammation and endothelial injury. Human umbilical vein endothelial cells (HUVECs) were cultured in vitro, serum-starved for 1 h at approximately 60% confluence, and stimulated with anti-β2GPI IgG (1:100 dilution) for 8 h. ICAM-1 protein expression was evaluated by Western blotting.
Results
Plasma Hcy levels were significantly elevated in Hcy-treated mice, whereas lipid levels did not differ among groups. Oil Red O staining demonstrated that hyperhomocysteinemia markedly promoted atherosclerotic plaque formation under a high-fat diet, while rituximab treatment significantly attenuated the pro-atherogenic effect of HHcy, indicating a role of B-cell–mediated immunity in HHcy-driven atherosclerosis. ELISA analysis showed that HHcy significantly increased plasma anti-β2GPI IgG levels in ApoE⁻/⁻ mice, whereas rituximab reduced HHcy-induced elevations of anti-β2GPI IgG, suggesting that HHcy promotes atherosclerosis through B cell–dependent production of anti-β2GPI antibodies. Immunofluorescence staining revealed enhanced co-localization of β2GPI with the endothelial marker CD31 following HHcy treatment, which was markedly reduced by rituximab administration. These findings indicate that HHcy induces endothelial β2GPI expression, facilitating binding of circulating anti-β2GPI IgG and promoting endothelial injury and plaque development. Consistently, anti-β2GPI IgG stimulation significantly activated HUVECs, as evidenced by increased ICAM-1 expression.
Conclusion
Our findings identify a previously unrecognized immunometabolic mechanism linking hyperhomocysteinemia to atherosclerosis, whereby HHcy promotes B cell–dependent production of anti-β2GPI IgG, leading to endothelial β2GPI upregulation, endothelial activation, and accelerated plaque formation. This study highlights an HHcy–B cell–autoantibody–endothelium axis as a key driver of vascular injury and provides mechanistic insight into immune-mediated atherogenesis.Oil Red O stainingElisa