Genome-wide changes conferred by trichostatin A, glucose and metformin in human vascular endothelial cells

Abstract

Endothelial dysfunction is a precursor of cardiovascular disease. It is characterised by the impaired production of the vasodilator nitric oxide and the development of a pro-thrombotic and pro-inflammatory endothelial state. Hyperglycaemia is a condition of elevated blood glucose levels as seen in diabetes and is a contributing factor to the development of endothelial dysfunction. The diabetes drug metformin reverses damage to the vascular endothelium, whereas trichostatin A (TSA), a prototypical HDAC inhibitor, has anti-inflammatory properties. While glucose, and TSA have been the focus of extensive research for decades, the genome-wide effects of these compounds in endothelial cells have not been adequately characterised.

Recent advances in high throughput sequencing (HTS) technologies have fast-tracked advancements in the genome-wide study of transcriptional regulation. The ability to look beyond single genes and loci is now readily accessible and affordable. This study utilised HTS to study genome-wide gene changes induced by hyperglycaemia and TSA in human endothelial cells. This genome-wide approach led to the identification of novel mechanisms of action by both compounds, while qRT-PCR was used to show that metformin attenuates changes in expression in hyperglycaemic endothelial cells.

HDAC inhibitors such as TSA, a diverse group of clinically relevant compounds, are thought to induce global histone hyperacetylation and therefore increased gene expression. In endothelial cells stimulated with TSA, the integration of gene expression changes (RNA-seq) with global histone acetylation data (ChIP-seq) identified that HDAC inhibition induces equal amounts of increases and decreases in gene expression and genome-wide histone acetylation. Histone deacetylation partially accounted for the anti-inflammatory effects of TSA. Deacetylation was dependent on the loss of histone acetyltransferases (HAT) binding at gene promoters. Indeed, the inhibition of structurally related HATs p300 and CBP prevented TSA-dependent reductions in expression at deacetylated genes such as the inflammatory cytokine IL6. A 50% reduction in CBP protein binding was also observed at the IL6 promoter. Transcription factors YY1, STAT2 and IRF3 were also associated with deacetylated promoters.

The use of HTS also identified novel pathways in hyperglycaemic endothelial cells that may be involved in the development of endothelial dysfunction. Hyperglycaemic endothelial cells expressed interferon-response pathway genes such as MX1 and IFI27. Transcription factor analysis implicates the activation of STAT1 and IRF1. Co-treatment of hyperglycaemic cells with metformin prevented glucose-dependent changes in gene expression, including interferon-response genes. Indeed, the effects of metformin in endothelial cells were dependent on glucose levels. In normoglycaemic cells, metformin subtly regulated changes in gene expression. In contrast, metformin was strongly associated with the reversal of gene expression changes induced by hyperglycaemia.

In this respect, metformin and TSA differ substantially in their anti-inflammatory properties. Metformin did not suppress pro-inflammatory gene expression in unstimulated endothelial cells, whereas TSA did. Interestingly, TSA and metformin were associated with the suppression of genes regulated by pro-inflammatory STAT and IRF family transcription factors.

In conclusion, the use of HTS facilitated the identification of novel responses in endothelial cells that were previously under-appreciated. This has provided new insights into the mechanism of action of HDAC inhibitors. It also suggests a potential mechanism for the development of endothelial dysfunction associated with hyperglycaemia and its reversal by metformin. The use of HTS has clear applications in attaining a deeper understanding of disease mechanisms as well as drug action, which, coupled together, have the potential to improve rational drug design.