Abstract Body (Do not enter title and authors here): BACKGROUND: Cellular senescence, an irreversible cell cycle arrest, has been associated with coronary heart disease (CHD). Senescent cells (SC) secrete proinflammatory molecules contributing to CHD. Insulin-like growth factor I (IGF1) protects against developing cell senescence and atherogenesis. IGF1 binding protein 7 (BP7) binds IGF1 and also interacts with IGF1 receptor (IGF1R), suppressing intracellular IGF1 signaling.
HYPOTHESIS: SC secrete BP7, and BP7 promotes cell senescence via suppression of IGF1 signaling.
METHODS: Cell senescence was induced by treatment of smooth muscle cells (SMC) with Bleomycin. Cell proliferation was quantified by live cell analysis system. BP7 levels were measured by Westerns in cell lysates and conditioned medium (CM) and by immunohistochemistry in atherosclerotic pigs. BP7/IGF1R binding was quantified by proximity ligation assay (PLA).
RESULTS: Bleomycin arrested SMC proliferation at 16h, upregulated senescence markers (beta-galactosidase and γH2A.X histone), and elevated BP7 levels in CM (3-fold increase, P<0.05) showing that SC secrete and upregulate BP7. We obtained CM from SC and used it to treat non-senescent SMC. CM inhibited IGF1-induced Akt phosphorylation in non-senescent cells, and CM incubation with BP7 antibody reversed this effect, indicating that BP7 mediates CM inhibitory effect on IGF1 signaling. BP7 overexpression (with pCMV-IGFBP7 vector) or inhibition of IGF1R (using Ganitumab, IGF1R antibody) suppressed cell proliferation, and upregulated senescence markers. BP7 overexpression also blocked IGF1-induced cell proliferation and migration (both are P<0.05) consisted with BP7 inhibitory effect on IGF1 signaling. Increased BP7 levels was detected in SMC in the fibrous cap (FC) in porcine plaque compared to BP7 level in vascular media (1.8-fold increase, P<0.05). BP7 upregulation in plaque correlated with elevated PLA signal indicating increased BP7/IGF1R binding. We used spatial transcriptomics to profile gene expression in the porcine atheroma. BP7 upregulation was detected in FC cluster (P<0.001) and it was co-localized with increased senescence module score.
CONCLUSIONS: SC upregulate BP7 and BP7 inhibits IGF1 signaling. Upregulated BP7 induces senescence in vitro and it was associated with SC in the advanced coronary plaque. Our results suggest a novel role of BP7 in senescence and atherogenesis and identify BP7 as a potential target for anti-atherogenic therapy.