IntroductionAtherosclerosis is a chronic arterial disease and a leading cause of morbidity and mortality worldwide.1 The arterial endothelium forms the first protective barrier against atherogenic stressors, but under sustained insult it can become dysregulated. Endothelial dysfunction is an early driver of plaque development, and endothelial cells (ECs) can even undergo phenotypic change into mesenchymal-like cells.2,3,4,5,6 This process, known as endothelial-to-mesenchymal transition (EndMT), has been shown to contribute to atherosclerosis by promoting EC dysfunction and plaque progression.7,8,9 In fact, cardiovascular risk factors and plaque microenvironmental stress (e.g., chronic inflammation, disturbed flow, and hypoxia) are known to trigger EndMT in ECs.Once initiated, EndMT leads to profound cellular changes that can exacerbate plaque development. EndMT-derived mesenchymal cells (often fibroblast-like) secrete extracellular matrix components and pro-inflammatory factors influencing plaque architecture and stability.7,10 Lineage-tracing and histopathology studies have confirmed that “transitioning” cells co-expressing endothelial and mesenchymal markers are present in atherosclerotic lesions. Notably, the extent of EndMT in plaques correlates with lesion complexity and instability.7 In advanced atherosclerotic plaques, a higher abundance of EndMT-positive cells is associated with fragile, unstable plaques prone to rupture.7 These findings underscore EndMT as a pathologic program that not only contributes to plaque growth but also potentially precipitates adverse clinical events.8,11Despite the growing recognition of EndMT in atherogenesis, the upstream mechanisms that link vascular risk factors to this endothelial phenotypic shift remain incompletely understood. Prior studies have largely focused on classical signaling pathways (such as TGF-β/SMAD, Notch, and BMP signaling) as drivers of EndMT.9,12,13,14,15,16 While these pathways activate transcription factors and epigenetic changes that initiate EndMT,6,17 much less is known about how the transcriptional machinery itself is mobilized during EndMT. Converting an EC to a mesenchymal cell requires coordinated upregulation of a broad gene program, suggesting that fundamental gene regulatory mechanisms are at play. In particular, the control of transcription at the RNAPII elongation stage has emerged as a crucial, albeit underappreciated, layer of gene regulation in dynamic cellular transitions.18Cells often prime critical genes for rapid activation by loading RNAPII at promoters in a paused state, allowing for prompt gene expression upon stimulation.19 This promoter-proximal pausing and subsequent release of RNAPII is a well-established mechanism to tightly control the timing and amplitude of transcriptional responses.18,20 Many stress-responsive and developmental genes are regulated at this pause-release checkpoint.19,21 For example, in resting immune cells, RNAPII initiates transcription of pro-inflammatory genes but stalls near the promoter, only resuming elongation when an inflammatory stimulus occurs.22,23 The transition from paused to actively elongating RNAPII is governed by the positive transcription elongation factor b (P-TEFb), a CDK9/cyclin T kinase complex that phosphorylates RNAPII and pause-inducing factors to enable productive elongation.20 Notably, P-TEFb does not act alone; it is recruited to target genes as part of large co-factor assemblies that integrate signaling cues with the transcriptional apparatus.20,21One of the key regulators of P-TEFb–mediated pause release is the super elongation complex (SEC). The SEC is a multiprotein complex assembled on scaffold proteins AFF1 and AFF4 (members of the AF4/FMR2 family), which tether P-TEFb to transcription sites and recruit additional elongation factors (such as ENL, AF9, and ELL proteins).20,24,25 Through this mechanism, the SEC serves as a master regulator of rapid transcriptional induction. Indeed, the AFF1/AFF4-containing SEC exhibits robust RNAPII C-terminal domain kinase activity and is uniquely required for the prompt activation of certain stress-responsive genes (for instance, only SEC is required for proper induction of the HSP70 heat shock gene upon stress).20 Genome-wide studies have further shown that AFF4/SEC plays a dominant role in facilitating immediate early gene expression in mammalian cells.26 The importance of SEC-mediated transcription elongation has been demonstrated in developmental biology and cancer pathogenesis, and CDK9 in particular is a target of current drug development, examples being BAY1143572/Atuveciclib (Bayer), PHA-767491 (Pfizer), LY2857785 (Eli Lilly) or AZ5576 (AstraZeneca),27 but its role in vascular disease has not been explored. To date, no studies have addressed whether this transcription elongation machinery contributes to endothelial dysfunction or atherogenesis.We hypothesized that atherosclerosis-relevant stress cues initiate endothelial-to-mesenchymal transition through a rapid shift in transcriptional kinetics, specifically RNA polymerase II pause release and processive elongation driven by the super elongation complex. To test this, we integrated human plaque transcriptomics, primary human endothelial cell transition models, genome-wide measurements of polymerase pausing and nascent transcription, and an in vivo hyperlipidemic atherosclerosis model. This multi-layered strategy allowed us to connect transcriptional elongation control to endothelial plasticity and plaque vulnerability across experimental and human disease contexts.ResultsThe components of the super elongation complex are increased by EndMT and in EndMT-positive ECs of human atherosclerotic plaque sectionsAtherosclerotic plaque and proximal adjacent vessel sections used as controls from the same patients were analyzed by single-cell RNA sequencing (scRNA-seq) (Fig. 1a–h). Atherosclerotic plaque samples showed increased mesenchymal markers such as TAGLN, FN1, FAP, and TGFB2, indicating EndMT-positive ECs (Fig. 1b). After cell clustering, the EC cluster was separated into EndMT-positive and EndMT-negative EC based on single-cell expression of EC and mesenchymal marker gene expression (Fig. 1c–f). The number of EndMT-positive ECs was increased in atherosclerotic plaque sections compared to control (proximal adjacent) vessel sections (Fig. 1e, f). Further separation of the EC transition process into an early EC-activated, a transitional signature, and a late EndMT signature showed components of the SEC activation as a hallmark of the transitioning endothelial state in human plaques (Fig. 1g–i). Expression of the SEC scaffolds AFF1 and AFF4 was reduced in fibroblasts, immune cells and smooth muscle cells of atherosclerotic plaque sections compared to control (proximal adjacent) vessel sections (Supplementary Fig. 1b). All SEC components were expressed in human cardiovascular tissues, such as left ventricle and atrium and in primary ECs isolated from human coronaries (Fig. 1j–l, Supplementary Fig. 1c). In human coronary artery ECs, EndMT was induced using 1-, 2- and 3-hit models that combine FGF2 loss, TGF-β and IL-1β treatment. This induction increased levels of SEC complex components at both the RNA and protein levels and was accompanied by a concomitant onset of endothelial dysfunction (Fig. 1m–w, Supplementary Fig. 1d–m).6 Together, our data indicate that SEC complex components are increased in EndMT-positive ECs of human atherosclerotic plaques.Fig. 1Full size imageThe SEC scaffold proteins AFF1 and AFF4 are expressed in human endothelial cells (ECs) and upregulated during the endothelial-to-mesenchymal transition (EndMT). a Single-cell sequencing data of human carotid artery plaques and proximal adjacent vessel sections used as control from the same patients (n = 3 per group). b Expression of EndMT signature genes in single-cell sequencing analysis from AP and CV tissue depicted in a dot plot (n = 3 per group). c Cell clustering of 51,117 cells from atherosclerotic plaque and control sections depicted in a UMAP plot. d Re-clustering of the EC cluster into EndMT-positive and EndMT-negative ECs in atherosclerotic plaques based on expression of EC markers PECAM1, CDH5, VWF, and EndMT markers TAGLN, FN1, FAP, and TGFB2 (depicted by a dot plot) was used as an EndMT signature score (n = 3 per group). e EndMT-positive and EndMT-negative ECs in atherosclerotic plaques (AP) (right) and proximal adjacent vessels section used as control (CV) (left). f Quantitative analysis of the mean percentage of EC (EndMTneg) and EndMT-positive cells in the EC cluster of AP and CV. g Schematic depiction of super elongation complex (SEC) composition built around the scaffold proteins AFF1 and AFF4. (h) Quantitative analysis of the mean percentage of quiescent EC, EC-activated, Transitional, and EndMT-positive cells in the EC cluster of AP and CV. (i–l) Expression of the SEC genes in ECs of human plaques and different human tissues and cardiovascular cell types. i SEC-associated gene expression in ECs (EndMTneg), EC-activated, Transitional, and EndMT-positive cells of carotid artery plaques and control vessel tissue depicted in a dot plot. j mRNA Expression of SEC-component genes in different human tissues as determined by semi-quantitative PCR. k AFF1-AFF4 expression in human umbilical vein endothelial cells (HUVECs), human carotid artery endothelial cells (HCAECs), human coronary artery smooth muscle cells (HCASMCs) and human cardiomyocytes (HCMs) determined by qPCR (n = 3). (l) Protein expression of SEC components in different human cardiovascular cell types. m EndMT in human ECs using a multi-hit strategy. n HCAECs were subjected to a 1-Hit (-FGF-2) or 2-Hit (-FGF-2/+TGF-β2) EndMT model, and gene expression was analyzed using bulk RNA sequencing (n = 4 per group). Venn diagram depicting upregulated genes in human coronary ECs undergoing EndMT. o Representative immunofluorescence staining of ECs under EndMT conditions with SM22 (red) and VE-Cadherin (green) and DAPI counterstain (blue) detected at 32X magnification. Scale bar represents 20 µm. Heatmaps depicting the expression of p EMT-related and q TGF-β receptor signaling pathway-related genes in human coronary ECs undergoing 1-Hit and 2-Hit EndMT vs. control ECs using RNA-seq (n = 4 per group). r AFF1 and s AFF4 expression in human coronary ECs undergoing EndMT (n = 4, One-way ANOVA, Dunnett’s multiple comparison test) as TPM using RNA-seq (n = 4/group). t Protein expression of EndMT-associated genes and u SEC components AFF1 and AFF4 in human coronary ECs (n = 4). v Quantification of Calponin and w AFF4 protein levels in EndMT-treated human coronary ECs (n = 5, One-way ANOVA, Dunnett’s multiple comparison test). CV – control vessel, AP – atherosclerotic plaque, Ctl – control. All bar graphs represent mean + SEM, * indicates P