Aims Histone H3 dimethylation at lysine 79 is a key epigenetic mark uniquely induced by methyltransferase disruptor of telomeric silencing 1-like (DOT1L). We aimed to determine whether DOT1L modulates vascular smooth muscle cell (VSMC) phenotype and how it might affect atherosclerosis in vitro and in vivo, unravelling the related mechanism. Methods and results Gene expression screening of VSMCs stimulated with the BB isoform of platelet-derived growth factor led us to identify Dot1l as an early up-regulated epigenetic factor. Mouse and human atherosclerotic lesions were assessed for Dot1l expression, which resulted specifically localized in the VSMC compartment. The relevance of Dot1l to atherosclerosis pathogenesis was assessed through deletion of its gene in the VSMCs via an inducible, tissue-specific knock-out mouse model crossed with the ApoE−/− high-fat diet model of atherosclerosis. We found that the inactivation of Dot1l significantly reduced the progression of the disease. By combining RNA- and H3K79me2-chromatin immunoprecipitation-sequencing, we found that DOT1L and its induced H3K79me2 mark directly regulate the transcription of Nf-κB-1 and -2, master modulators of inflammation, which in turn induce the expression of CCL5 and CXCL10, cytokines fundamentally involved in atherosclerosis development. Finally, a correlation between coronary artery disease and genetic variations in the DOT1L gene was found because specific polymorphisms are associated with increased mRNA expression. Conclusion DOT1L plays a key role in the epigenetic control of VSMC gene expression, leading to atherosclerosis development. Results identify DOT1L as a potential therapeutic target for vascular diseases. Key question Epigenetics plays an important role in the regulation of atherosclerosis development. Its role in vascular smooth muscle cell biology and immune cell recruitment is not yet fully understood. Key finding Inhibition of the epigenetic enzyme disruptor of telomeric silencing 1-like (DOT1L) in vascular smooth muscle cells significantly reduces atherosclerosis progression, directly modulating Nfκb1 and Nfκb2 transcription. These genes are master regulators of inflammation, able to induce the expression of CCL5 and CXCL10 cytokines, which play a fundamental role in atherosclerosis development. Take home message DOT1L could be a promising therapeutic target since its inhibition reduces plaque progression.

Intimal hyperplasia is the leading cause of graft failure in aortocoronary bypass grafts performed using human saphenous vein (SV). The long-term consequences of the altered pulsatile stress on the cells that populate the vein wall remains elusive, particularly the effects on saphenous vein progenitors (SVPs), cells resident in the vein adventitia with a relatively wide differentiation capacity. In the present study, we performed global transcriptomic profiling of SVPs undergoing uniaxial cyclic strain in vitro. This type of mechanical stimulation is indeed involved in the pathology of the SV. Results showed a consistent stretch-dependent gene regulation in cyclically strained SVPs vs. controls, especially at 72 h. We also observed a robust mechanically related overexpression of Adhesion Molecule with Ig Like Domain 2 (AMIGO2), a cell surface type I transmembrane protein involved in cell adhesion. The overexpression of AMIGO2 in stretched SVPs was associated with the activation of the transforming growth factor β pathway and modulation of intercellular signaling, cell-cell, and cell-matrix interactions. Moreover, the increased number of cells expressing AMIGO2 detected in porcine SV adventitia using an in vivo arterialization model confirms the upregulation of AMIGO2 protein by the arterial-like environment. These results show that mechanical stress promotes SVPs' molecular phenotypic switching and increases their responsiveness to extracellular environment alterations, thus prompting the targeting of new molecular effectors to improve the outcome of bypass graft procedure.

Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Ricerca Corrente Introduction We previously demonstrated that mechanical stress deriving from coronary flow/pressure patterns in the human saphenous vein (SV) conduits induce release of Thrombospondin-1 (TSP-1) by smooth muscle cells, and this activates adventitial progenitors (SVPs) pathologic activation. Purpose In this study, we show a cooperation of the TGF-β/TSP-1 signaling with the mechanically-activated Hippo transcriptional pathway in fibrotic SVPs commitment. Methods Human derived-SVPs were isolated using a MACS based protocol with a positive selection for CD34 and a negative depletion of CD31+ cells. We performed an RNA-seq analysis of SVPs subjected to 10% uniaxial deformation (n=5), followed by differential gene expression and pathway analyses. We validated results in vitro and in two animal models of vein arterialization. Results A response of SVPs to mechanical stimulation was assessed from variations in cell alignment, circularity and area. The susceptibility of SVPs to uniaxial strain was revealed by a trend of the cells to orientate in orthogonal direction to the strain field and changes in cell shape. Mechanically stimulated cells for 72 hrs showed a significant increase in their motility as verified by migration assays in the presence of medium supplemented with 10% serum. RNA-seq analysis of the total transcriptome expressed in these cells with/without mechanical stimulation was performed. The differentially expressed genes (DEGs) analysis highlighted a maximum variation of the transcriptome at 72hrs of mechanical stimulation vs. static controls with n=819 DEGs. A gene enrichment analysis revealed an involvement of the HIPPO/YAP/TEAD and of the TGF-β/SMAD transcriptional circuitries in mechanically-stimulated cells. In keeping, immunofluorescence and RT-qPCR showed an increase in YAP nuclear translocation and activity. We treated cells with a cytoskeleton inhibitor (Forskolin, FRSK) and a drug (Verteporfin, VTP) that prevents the interaction of the YAP/TAZ complex with TEADs. Both drugs inhibited expression of YAP-transcriptional targets and cellular motility in response to serum. We then treated cells with TGF-β1, TSP-1 alone or in combination. Under these conditions we observed an increased expression of YAP targets and CollA1, a higher amount of Collagen secretion in the supernatant and a higher association of YAP with pSMAD3. All these effects were blunted by VTP. YAP nuclear localization was finally validated in two models of vein arterialization in mice and pigs. Conclusions Our data suggest a convergent activation of Hippo/TGF-β pathways in the failure of the aorto-coronary bypass and highlight a future novel strategy to limit its progression in patients.