Introduction: Mounting evidence indicates that the process of endothelial-to-mesenchymal transition (EndMT) contributes to the initiation and progression of atherosclerosis. Stress stimuli induce a phenotypical change of endothelial cells (ECs) towards mesenchymal-like cells, with loss of endothelial cell markers and increased expression of mesenchymal genes. EndMT is based on rapid changes in the gene expression program of the stressed cells, which is mediated by the RNA polymerase II (RNAP II). AFF1, as part of the super elongation complex, regulates RNAP II pause and release, as well as the transcriptional elongation speed. Therefore, we aimed to analyze the potential contribution of RNA elongation to the process of EndMT. 

 

Methods and Results: AFF1 expression was elevated in human atherosclerotic plaques (N=32) compared to normal arteries (N=32) (P<0.05). Therefore, we characterized the expression profile of all AFF family members in primary human vascular cells. AFF1 and AFF4 exhibited considerable expression levels in human coronary artery endothelial cells (HCAEC), whereas the two other family members, AFF2 and AFF3, showed only modest levels. After induction of EndMT in ECs, AFF1 and AFF4 were significantly increased (1.9-fold and 2.65-fold, respectively. The expression of AFF1 and AFF4 can be reduced on protein level using two established small molecule inhibitors. The inhibition of AFF1/AFF4 reduced the induction of EndMT as measured by the reduction of the mesenchymal markers SM22 (-87.58% ± 2.78%; p<0.05) and calponin (-93.76% ± 0.81%; p<0.05), while the EC marker LYVE1 was not changed. We found AFF1 and AFF4 interacting with the promotor site of the EndMT marker calponin. Proximal promoter pausing is indicated by enrichment of phosphorylated serine 5 at the carboxyterminal domain of the RNAP II (CTD, pSer5). Indeed, using ChIP we found the proximal promoter of calponin to be enriched with phosphorylated-RNAP II (CTD, pSer5) indicating promoter pausing of stress gene expression such as calponin in physiological ECs.  

Conclusion: The RNA polymerase II elongation factors AFF1 and AFF4 are increased in human atherosclerotic plaques. AFF1 and AFF4 are co-enriched with paused polymerase II at promoters of EndMT stress genes, such as calponin. Inhibition of AFF1 and AFF4 using two small molecules resulted in reduced induction of EndMT. These results suggest an important function of RNA elongation in the rapid gene response to stress stimuli contributing to EndMT.