*Purpose: Endothelial cells (EC) centrally regulate leukocyte access to peripheral tissue and immune cell activation, and exhibit remarkable tissue-specific diversity. However, the molecular mechanisms underlying the heterogeneity of vascular inflammation are largely unknown, and consequently there is a lack of therapies that specifically target the endothelium in transplant rejection. We recently reported that the transcriptional regulator BCL6 was significantly enriched in cardiac EC. In this study, we asked whether the transcription factor B-cell lymphoma 6 (BCL6) is a modifier of inflammation and apoptosis driven by NFκB in EC.

*Methods: Using pharmacological inhibitors of BCL6 (79-6, FX1, BI-3812, BI-3802) and siRNA in primary human endothelial cells (EC), the impact of BCL6 on TNFα-induced NFκB activity, adhesion molecule and chemokine expression, and global transcriptome changes, was measured with an NFκB-luciferase reporter, flow cytometry, Luminex, and RNA-Seq, and re-analysis of public Molecular Microscope datasets of cardiac allograft rejection, and ENCODE ChIP-Seq data of the BCL6 cistrome.

*Results: BCL6 basal expression was found among endothelial cells types in diverse organs, being particularly enriched in the cardiac endothelium in mouse and human. Interestingly, BCL6 mRNA was upregulated in human transplanted hearts with rejection and injury, compared with clinically normal grafts. Pharmacological agents that target BCL6 significantly altered the endothelial response to TNFα, predominantly NFκB-driven targets. Delving in further, we found that BCL6 DNA binding motifs are highly similar to NFκB consensus motifs and appear to overlap in a tiled manner. In ChIP-Seq data, BCL6 bound directly to numerous endothelial inflammatory genes, including VCAM1, CXCL2, CX3CL1, SELE, and CCL5.Moreover, NFκB and BCL6 BTB domain inhibitors FX1 and BI-3812 suppressed, while BCL6 depletion potentiated, activation of endothelium by TNFα, including 1) NFκB transcriptional activity, 2) chemokine production (GROα, IL-6, MCP-1), 3) adhesion molecule expression (VCAM-1), and 4) binding of allogeneic leukocytes. Similarly, the pharmacological BTB domain inhibitors of BCL6 and NFƙB decreased apoptosis under TNFα stimuli, promoting EC viability.

*Conclusions: Our results show that BCL6 regulates NFκB-dependent transcription in endothelium, potentially through chromatin remodeling. We conclude that BCL6 is involved in the initiation, magnitude and termination of inflammation and is implicated in the apoptosis response driven by NFκB in cardiac endothelial cells, and may be a therapeutic target to ameliorate vascular inflammation in transplant rejection.

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