Phosphatidic Acid Mediated Sonodynamic Therapy Facilitates M1 Macrophage Osteoclastic Function and Alleviates Early Vascular Calcification

BACKGROUNDVascular calcification significantly influences the onset and outcome of cardiovascular events, yet no effective treatment currently exists. Dysfunction of osteoclastic macrophages contributes to the formation of calcification. Our previous studies have shown that sonodynamic therapy (SDT) can rapidly reverse atherosclerotic plaques by targeting macrophages. This study aimed to investigate the effect of SDT on reducing early or mild vascular calcification by modulating the function of osteoclastic macrophages. METHODSThirty-two patients with symptomatic femoropopliteal peripheral artery disease (PAD) were recruited to evaluate changes in vessel CT values and the target-to-background ratio (TBR) using positron emission tomography/computed tomography (PET/CT) 30 days post-SDT. An early calcification model was established in ApoE-/- mice, followed by SDT intervention. Frozen plaque sections from the mice were collected for mass spectrometry imaging (MSI)-based spatial metabolic analysis in situ. The NHGRI-EBI GWAS Catalog database and the human single-cell eQTL database (scQTLbase) were employed to analyze the causal relationship between key enzyme genes involved in phosphatidic acid (PA) synthesis in macrophages and vascular calcification using two-sample Mendelian randomization. To investigate cell ossification, calcification, and underlying mechanisms, RAW264.7 mouse macrophages were treated with a medium containing receptor activator of nuclear factor kappa-B ligand (RANKL), while mouse aortic vascular smooth muscle cells (MOVAS cells) were exposed to a calcification medium. RESULTSSDT significantly reduced the number of mildly calcified sites and the target-to-background ratio (TBR) of these sites in patients with femoropopliteal peripheral artery disease (PAD). In ApoE-/- mice, SDT alleviated early calcification of atherosclerotic plaques. MSI revealed that SDT altered the composition and distribution of lipid metabolites in atherosclerotic plaques, notably increasing the content of PA in the early calcified regions. Analysis of single-cell sequencing databases showed that key enzyme genes involved in PA synthesis--PLD1, PLD3, AGPAT4, and diacylglycerol kinase E (DGKE)--were enriched in macrophages of human coronary artery plaques. Mendelian randomization analysis indicated that DGKE negatively regulated coronary artery calcification. In vitro studies demonstrated that PA mediates SDT to promote M1 macrophage fusion and enhance carbonic anhydrase II (CA2) expression, thereby improving osteoclastic function and alleviating early calcification of MOVAS cells via the reactive oxygen species (ROS)-DGKE-PA pathway. In vivo, the CA2 inhibitor acetazolamide impaired the effects of SDT and exacerbated early calcification of atherosclerotic plaques in ApoE-/- mice. CONCLUSIONThis study demonstrates that PA-mediated SDT promotes M1 macrophage fusion and CA2 expression, improving osteoclastic function and alleviating early calcification through the ROS-DGKE-PA pathway. REGISTRATIONURL: https://www.clinicaltrials.gov; Unique identifier: NCT03457662. What Is New?1. SDT reduces early calcification in patients with symptomatic femoropopliteal PAD and in ApoE-/- mouse models of early calcification. 2. SDT upregulates the expression of PA in the early calcified regions of ApoE-/- mouse models. 3. PA-mediated SDT facilitates M1 macrophage fusion and enhances CA2 expression, thereby improving osteoclastic function and alleviating early calcification through the ROS-DGKE-PA pathway. What Are the Clinical Implications?1. Targeting M1 macrophage DGKE-PA may serve as a potential intervention for treating early vascular calcification. 2. The combination of MSI and Mendelian randomization analysis proves to be an effective method for exploring key signaling lipids in disease. 3. PA-mediated SDT represents a promising approach for the effective reduction of early vascular calcification.