Introduction

During endothelial-to-mesenchymal transition (EndMT), differentiated adult endothelial cells (EC) transform into mesenchymal-like cells, resulting in the loss of their endothelial functions such as the maintenance of vascular integrity. EndMT was detected in unstable (vulnerable) and ruptured atherosclerotic plaques. Various stress stimuli, including hypoxia, oxidative stress, or inflammation, induce EndMT and lead to adaptive gene expression. During cell and tissue stress, the cold shock proteins YBX1 and YBX3 are regulated and have been implicated in inflammation and fibrosis. Therefore, we analyzed the members of the YBX family in the process of EndMT.

Methods and Results

The mRNA expression of YBX1 and YBX3 in human EC was higher than the expression of the prototypical EC gene VE-cadherin, whereas YBX2 was absent (n = 3-7). Analysis of a human tissue panel revealed YBX1 and YBX3 expression in the left ventricle and left atrium. Induction of EndMT using human primary EC resulted in an increased expression of YBX1 compared to control cells (n=6; p<0.05). Next, we performed Single Cell Sequencing after EndMT. EC were divided based on expression of the mesenchymal marker. The expression of YBX1 and YBX3 was increased in the EndMT-positive cell fraction (EndMT-: n=4274, EndMT+: n=2469, p<0.05). Spatial Transcriptomics of vascular sections from the brachiocephalic artery of Ldlr^-/-  mice fed a high-fat diet showed an increase of Ybx1 and Ybx3 expression in the plaque area compared to clusters of vascular section from wild-type mice (p < 0.05).

YBX1 and YBX3 are RNA binding proteins. RNA immunoprecipitation showed that YBX1 and YBX3 interact with key mRNAs of the EndMT process, such as TGF-β1 (641 and 550 fold vs IgG, respectively) and TGF-β2 (3030 and 1803 fold vs IgG, respectively). The pooled siRNA-based knockdown of YBX1 and YBX3 resulted in selective inhibition of components of the EndMT process during EndMT induction. Interestingly, knockdown of YBX1 resulted in a 95% reduction of the EndMT marker VCAN compared to EndMT (p<0.05), while knockdown of YBX3 had no effect on VCAN induction during EndMT. In contrast, knockdown of YBX3 resulted in a 70% reduction of the EndMT marker SM22 and a 50% reduction of TGF-β2 compared to EndMT, while knockdown of YBX1 had no effect on SM22 induction during EndMT. These findings suggest an inhibition of the positive feedback loop of TGF-β2 by the YBX genes.

Discussion

The cold shock proteins YBX1 and YBX3 are upregulated during endothelial-to-mesenchymal transition and in atherosclerotic plaques. YBX1 and YBX3 interact with mRNAs of the TGF-β signaling pathway and YBX genes modulate key components of the EndMT process.