Abstract Background The transcriptional response to environmental changes, such as nutrient availability, can unfold over hours or days through transcription factors and chromatin modifications. In contrast, the response to stress stimuli, such as pathogens, occurs within minutes via RNA elongation, as seen in the inflammatory response. Prolonged stress stimuli such as inflammation, TGF-β or hypoxia induce endothelial dysfunction characterized by endothelial-mesenchymal transition (EndMT), a process in which endothelial cells (EC) transform into mesenchymal cells and lose their EC properties. EndMT-positive cells are found in vulnerable atherosclerotic plaques, and EndMT is thought to play a role in the development and progression of atherosclerosis. The super elongation complex (SEC), consisting the proteins AFF1 and AFF4, controls RNA elongation. We aimed to investigate the role of RNA elongation in the initiation and progression of EndMT and atherosclerosis. Methods and Results Single-nucleus sequencing of human coronary artery plaques revealed a higher number of AFF1/AFF4 EndMT double-positive cells compared to vascular control tissue. RNA elongation rate increases as early as 10 minutes after EndMT induction, as shown by sequencing of newly synthesized 4sU-labelled RNA. The rapid induction of gene expression depends on the release of paused RNAPII present at the promoter site. ChIP-sequencing revealed a reduced occupancy of RNAPII at promoter sites in EndMT, suggesting a rapid pause release of RNAPII during EndMT. Treatment with a new specific chemical inhibitor of RNA elongation, which destabilizes the SEC and thereby leads to degradation of the AFF1/AFF4 protein, also reduced EndMT in primary human EC. In addition, inhibitor treatment reduced EndMT-induced RNA elongation rates, indicating a regulatory contribution of RNA elongation in EndMT. Treatment with the chemical inhibitor in an atherosclerotic mouse model (PCSK9-AAV8 + HFD for 20 weeks) reduced the area of atherosclerotic lesions as shown by en face aortic staining and aortic root analysis. In addition, inhibitor treatment reduced plaque protrusion, the extent of macrophage infiltration and the number of EndMT-positive cells in the atherosclerotic plaques. Conclusions RNA elongation increased immediately after induction of EndMT. Treatment with a novel inhibitor of the SEC reduced EndMT, plaque size, plaque protrusion and macrophage burden in an atherosclerotic mouse model. These data suggest that RNA elongation contributes to both EndMT and early atherosclerosis. This opens up a whole new field of potential targets for future therapeutic approaches in atherosclerotic cardiovascular diseases.
Kokot et al. (Sat,) studied this question.