*Purpose: Histone modifications such as acetylation control gene expression. Histone deacetylases (HDACs) have been proposed as an attractive therapeutic approach to counteract detrimental gene expression, in transplant and other inflammatory diseases. We aimed to characterize the role of HDACs in endothelial pro-inflammatory NFκB responses.

*Methods: HDAC activity was measured in primary human aortic endothelial cells (EC) stimulated with TNFα (20ng/mL) using a fluorescent substrate. EC were pre-treated with the HDAC inhibitor TSA (20μM, 30min) and left unstimulated or treated with TNFα (20ng/mL) for 4hr or 24hr. mRNA was measured by RNA-Seq, and gene expression modules analyzed by gene ontology; protein expression was measured by flow cytometry and ELISA.

*Results: Endothelial cells treated with TNFα transiently increased HDAC activity at 4hr (151.3±16.0% of baseline), which was reduced to baseline at 24hr. Treatment with TSA abrogated HDAC activity, as expected (37.6±1.7% of baseline).Pre-treatment of EC with TSA prior to short-term (4hr) TNFα reduced CX3CL1, CXCL8, CXCL6, CCL2, TNFSF10 but not the early induction of CXCL2, CXCL1, CCL5, SELE. On the other hand, early expression of CCL20 and ICAM1 was augmented. Most long-term (24hr) TNFα effects were significantly blocked by TSA. Pro-inflammatory gene induction, including CXCL8, VCAM1, IL6, CSF1 was reduced. However, no effect on ICAM1, CCL5, HLA-A; NFKBIA, NFKB2, TNFAIP3 expression was observed, and enhanced BST2, CCL20, TNFRSF9 expression resulted. Surprisingly, although TNFα alone has no effect on HLA class II expression, the combined effect of TSA+TNFα increased HLA-DPB1, DQB1, DMA/B. Importantly, TSA treatment alone dramatically altered gene expression patterns in endothelium. 869 genes were highly upregulated (>5-fold), and 883 genes were down-regulated (<0.3-fold). Down-regulated genes were enriched for GO terms like “regulation of chromatin binding,” and “blood vessel maturation”. Enriched modules among up-regulated genes included “animal organ development” and “cell morphogenesis.” By 4hr, the master endothelial regulator ERG was significantly decreased, which was followed at 24hr by loss of endothelial marker and angiogenic genes (FLI1, ICAM2, PECAM1, S1PR1, MMRN1, ECSCR, EMCN, NOS3, NOTCH4, CDH5, KDR). Conversely, TSA upregulated mesenchymal and developmental genes (4hr: KLF2, WNT4, WNT5A; 24hr: NOTCH3, EPHB3, MEIS3, WNT2B, SNAI1, SNAI2, COL1A1, PDGFR, SOX8).

*Conclusions: HDAC inhibition potently attenuates much of the NFκB-induced inflammation that may be relevant for acute rejection mechanisms. However, broad HDAC inhibition also promotes a loss of endothelial cell specification indicative of an endothelial-to-mesenchymal transition. These results highlight the potential adverse effects of using epigenetic modifiers as immunosuppressants in transplantation. Further, they underscore the protective role for HDACs in maintenance of endothelial identity, the loss of which is implicated in vasculopathy.

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