Gene expression of HUVECS treated with either siRNA to CTRL, PDHA1, or SREBF2 and treated with TNF 16 hours

The endothelium is a major target of the proinflammatory cytokine, tumor necrosis factor alpha (TNFa). Exposure of endothelial cells (EC) to proinflammatory stimuli leads to an increase in mitochondrial metabolism, however, the function and regulation of elevated mitochondrial metabolism in EC in response to pro-inflammatory cytokines remains unclear. Using high-resolution metabolomics and 13C-glucose labeled flux techniques, we demonstrate that pyruvate dehydrogenase activity (PDH) and tricarboxylic acid cycle flux is elevated in human umbilical vein ECs (HUVECs) in response to overnight (16 hrs) treatment with TNFa (10 ng/mL). Mechanistic studies indicate that TNFa mediates these metabolic changes via mitochondrial specific protein degradation of pyruvate dehydrogenase kinase 4 (PDK4, inhibitor of PDH) by the Lon protease via an NF-?B dependent mechanism. Using RNA sequencing following siRNA mediated knockdown of the catalytically active subunit of PDH, PDHE1a (PDHA1 gene), we show that PDH flux controls the transcription of approximately one-third of the genes that are upregulated by TNFa stimulation. Notably, TNFa induced PDH flux regulates a unique signature of proinflammatory mediators (cytokines and chemokines) but not inducible adhesion molecules. Using metabolomics and ChIP sequencing (H3AcK27), we demonstrate that TNFa induced PDH flux promotes histone acetylation of specific gene sets via citrate accumulation and ATP-citrate lyase activity. Together, these results indicate that targeting endothelial glucose oxidation and/or acetyl CoA generation may offer a novel therapeutic strategy in the treatment of vascular inflammation. Overall design: Comparative gene expression of early passage HUVECs treated with either siRNA to CTRL, PDHA1, or SREBF2 and treated with TNF 16 hours.