Abstract 627: Combined Plaque Evaluation by Ultrasound and Microarrays Reveals Bclaf1 as a Novel Regulator of Smooth Muscle Cell Transdifferentiation in Atherosclerosis

Abstract

Objective: Understanding molecular processes behind carotid plaque instability is necessary to develop methods that can identify patients and lesions at risk of stroke. Here, we investigated molecular signatures in human plaques stratified by echogenicity as assessed by duplex ultrasound (US). Results: Plaque echogenicity measured by US was correlated to microarray profiles from lesions retrieved at surgery (n=96). Pathway analyses highlighted enrichment of cell apoptosis and proliferation, and BCLAF1 (BCL2 associated factor 1) as the most significantly dysregulated gene (adjusted p<0.0001). BCLAF1 was strongly downregulated in plaques vs. control tissues, positively correlated to markers of cell proliferation and negatively to apoptosis, at both transcriptomic and proteomic level. Immunohistochemistry showed that BCLAF1 was localized in smooth muscle cells (SMCs) nuclei and repressed early during atherogenesis, but reappeared in CD68+ cells in advanced plaques. Proximity ligation assay demonstrated interaction of BCLAF1 with previously reported interaction partners THRAP3 and BCL2, in normal arteries and plaques. In vitro , stimulation of SMCs with pro-survival factors EGF, bFGF, PDGFB resulted in induction of BCLAF1, while it was suppressed by macrophage-conditioned medium. Moreover, BCLAF1 silencing in SMCs led to downregulation of BCL2 and SMC markers, and a decrease in proliferation and adhesion (p<0.0001). Transdifferentiation of SMCs using oxLDL, confirmed by CD68 upregulation and MYH11 repression, was accompanied by upregulation of BCLAF1. However, a combination of oxLDL exposure and BCLAF1 silencing, resulted in preserved expression of MYH11 and prevented transdifferentiation. Finally, BCLAF1 expression in CD68+/BCL2+ cells of SMC origin, was verified in plaques from MYH11-lineage tracing atherosclerotic mice. Conclusions: Carotid plaque echogenicity correlated with enrichment of molecular pathways associated with cell survival and apoptosis and identified BCLAF1, previously not described in atherosclerosis, as the most dysregulated gene. Functionally, BCLAF1 appeared to promote SMC survival by transdifferentiation into macrophage-like phenotype, by interacting with BCL2 and THRAP3.