Intracellular Osteopontin of Macrophages Promotes Carotid Plaques Formation by Inducing Foam Cells and Releasing Proinflammatory Cytokines

Background Atherosclerotic carotid plaque is the main cause of cerebrovascular diseases, but the molecular and cellular mechanisms involving in carotid plaque formation have not yet been fully uncovered. Methods Single cell RNA sequencing of human carotid plaques was used to identify major cell types. Cell‐cell communication and machine learning analyses were used to determine the key cell cluster involved in plaque development. In vitro experiments were conducted to explore the role of SPP1 (osteopontin) in foam cells formation. Clinical and histological analyses were also performed to validate the involvement of osteopontin in atherosclerosis. Results Eight distinguishable major cell types were identified in atherosclerotic carotid tissue. Cell‐cell communication and machine learning analyses identified SPP1 hi macrophage as the key cell cluster in plaque development, and functional annotation further highlighted the involvement of hypoxia response and cholesterol metabolism. Culturing mouse macrophages under 1% O 2 hypoxia condition with the addition of oxidized low‐density lipoprotein (ox‐LDL) significantly induced membrane translocation of CD36 and SR‐A, enhanced the expression of SPP1 (osteopontin), promoted foam cells formation, and activated NF‐κB pathway to release proinflammatory cytokines. All these changes of macrophages under hypoxia and ox‐LDL stimulations were effectively mitigated by inhibiting intracellular osteopontin. Clinical data and histological analysis of surgical resected carotid plaque tissue samples also confirmed the role of osteopontin in atherosclerosis. Conclusions Intracellular osteopontin of macrophages promotes plaque formation by inducing foam cells and releasing proinflammatory cytokines. Our data provide novel mechanistical insight and therapeutic strategy on atherosclerotic carotid plaque.