Abstract Body: Introduction: Atherosclerosis, a major cause of cardiovascular disease, develops when cholesterol-rich plaques build up in the arteries, causing narrowing of the artery lumen. Consequently, atherosclerosis is an inflammatory disease that involves endothelial cell (EC) and macrophage (MP) dysfunction, leading to abnormal lipid uptake into blood vessels. Cysteinyl leukotrienes (cys-LTs; LTC₄, LTD₄, LTE₄) are inflammatory molecules that act through their receptors, CysLT₁R and CysLT₂R. In our previous studies, we demonstrated that cys-LTs enhance ox-LDL uptake in bone marrow-derived macrophages (BMDMs) via cysLT₁R. While limited studies have examined the role of cys-LTs in ECs or MPs individually, their effects on EC-MP interactions remain unclear. Therefore, we investigated the role of CysLTR signaling in EC-MP interactions using an in vitro co-culture system and an in vivo PCSK9-induced atherosclerosis model employing CysLTR knockout mice.
Hypothesis: CysLTR signaling augments EC-MP interactions and promotes atherosclerosis progression.
Methods: In vitro, mouse ECs were co-cultured with BMDMs from WT, Cysltr1^-/- and Cysltr2^-/- mice in transwell system for 6 hours. Supernatants and cells were analyzed for ELISA and qPCR respectively. In vivo, atherosclerosis was induced in male and female mice by ip injections of PCSK9-AAV followed by high-fat diet (HFD) for 12 weeks (n=48). Aortic plaques were examined to analyze plaques and their composition using Oil-red O staining and immunofluorescence.
Results: In EC-BMDM co-cultures, BMDMs upregulated CysLT₁R and displayed an inflammatory phenotype, with increased levels of IL-6, IL-1β, GM-CSF, and scavenger receptors like OLR-1, compared to isolated cultures. Similarly, ECs co-cultured with BMDM showed increased expression of adhesion molecules like E-selectin, ICAM-1, and VCAM-1 at the transcript level. Interestingly, Cysltr1^-/- BMDMs co-cultured with ECs exhibited significantly reduced levels of inflammatory cytokines and scavenger receptors compared to WT BMDMs Importantly, PCSK9 + HFD-treated Cysltr1^-/- mice exhibited reduced plaque formation compared to PCSK9 + HFD treated WT mice. Further, we found reduced macrophage infiltration and smooth muscle cell migration in plaque areas of Cysltr1^-/- mice compared to WT mice.
Conclusion: Our findings reveal that CysLT₁R plays a key role in regulating EC-MP interactions and driving atherosclerosis progression, highlighting it as a potential therapeutic target.