Abstract Background Atherosclerosis is a disease characterized by lipid-rich, immune-infiltrated plaques in arteries that cause cardiovascular events (e.g. heart attack) upon rupture. In atherosclerotic plaques, smooth muscle cells (SMCs) proliferate and undergo phenotypic transitions. Lineage tracing of SMCs has shown that SMCs can lose contractile markers and express markers of other cell types, such as macrophages. These phenotype-switched SMCs were demonstrated by our group to upregulate a calcium-binding alarmin protein, S100A4, and S100A4 neutralization using an antibody reduced plaque burden in a murine model of the disease. Extracellular S100A4 can induce pro-inflammatory signalling in SMCs through Toll-like receptor 4 (TLR4), while intracellular S100A4 can modulate cell motility via interaction with the cytoskeleton in several cell types, including macrophages and cancer cells(1). S100a4 is expressed in a plurality of immune cells that are also detected in atherosclerotic plaques, such as monocytes, dendritic cells, and T cells(1). However, the role of the immune-derived S100A4 pool is not well characterized in atherosclerosis. Moreover, the mechanism of S100A4 release into the extracellular space is poorly understood(1). Another protein of the S100 family, S100A8/A9, was recently shown to be unconventionally secreted downstream of the activation of NLR family pyrin domain containing 3 (NLRP3) inflammasome(2). Purpose This project aims to address the role of S100A4 in the immune compartment in atherosclerotic plaques and to investigate the unconventional release of S100A4 via the NLRP3 inflammasome. Methods S100A4 was depleted in the circulating immune compartment of the S100A4+/+ ApoE-/- atherosclerosis mice model (S100a4 WT) by bone marrow transplantation from S100A4-/- ApoE-/- mice donors (S100a4 KO). After a high cholesterol diet, plaque development was assessed in the aorta using Oil Red O and at the aortic root. To streamline and standardize the quantification of aortic root cryosection immunofluorescence images, a semi-automated, AI-assisted image analysis pipeline was developed using Segment Anything Model 2 (Meta Inc.). To understand whether NLRP3 is involved in S100A4 release from SMCs, SMC cultures were treated with NLRP3 stimulants (lipopolysaccharide and nigericin). Results In mice receiving S100a4 KO bone marrow cells, atherosclerotic plaque surface area in the aorta was reduced from 9,4% (± 0.92) of the aorta to 6,5% (± 0,73; p = 0.0226). Preliminary results from SMCs treated with NLRP3 stimulants suggested the expected nuclear translocation of transcription factor NFκB and the upregulation of some inflammasome-related genes (Tlr4, Nlrp3), but not others. Conclusions The depletion of S100A4 protein in circulating immune cells reduced atherosclerotic plaque surface area in the aorta in our preliminary results. The SMCs are partially responsive to stimulants of the NLRP3 inflammasome, but NLRP3 activity needs to be confirmed.For image description, please refer to the figure legend and surrounding text.
Xu et al. (Fri,) studied this question.