Histone Deacetylase Inhibitors Suppress Transcription of NADPH Oxidases and Ameliorate Pulmonary Hypertension
Authors: Haigh Stephen Barton , Chen Feng , Li Xueyi , Meadows Mary Louise , Zhou Jiliang , Stepp David , Weintraub Neal , Barman Scott , Fulton David 
Published: November 2023

Excessive production of reactive oxygen species (ROS) have been shown to be detrimental in cardiovascular disease as well as many other conditions such as cancer and neurodegenerative diseases. There are many sources of ROS, but the NADPH Oxidases (NOX) are considered to be a major contributor to increased oxidant tone and contribute to inflammation, hyperproliferation and apoptosis. The NOX enzymes are a family of transmembrane oxidoreductases that are dedicated to the production of superoxide and other ROS. Pulmonary hypertension (PH) is a rare condition resulting from progressive increases in blood pressure and proliferative changes in the pulmonary artery that leads to right ventricular failure and death. Unlike some vascular disorders the cause of PH is poorly understood. Previous studies have shown that both NOX expression and ROS levels are increased in PH. The mechanisms regulating NOX expression are incompletely understood and particularly so in the context of PH. Therefore, the goal of our study was to investigate the role of HDAC and epigenetics in regulating NOX enzyme expression, ROS levels and vascular remodeling in PH. Inhibition of HDACs in macrophages, THP1 and HL‐60 cells resulted in decreased levels of ROS as well as decreased expression of NOX2 at both the protein and RNA levels. Inhibition of HDACs in other cell types including vascular cells (endothelial, fibroblast and smooth muscle) as well as cancer cells (cacao, PC3) also resulted in the consistent down regulation of NOX enzyme expression. Mechanistically, HDAC inhibitors would be expected to increase the acetylation of histones, promote transition from heterochromatin to euchromatin and increase the accessibility of gene promoters to enable increased gene expression. As we observed the opposite results on NOX expression we next assessed the epigenetic modifications of histones in close proximity to the proximal promoter regions of NOX genes using ChIP. Surprisingly we found reduced histone acetylation (H3K9ac) at the proximal promoter for NOX2 but this was consistent with reduced H3K4me3 and RNApol2 in the same region. To functionally explore whether HDAC inhibitors reduced chromatin accessibility, we targeted inactive CAS9 linked to transcriptional activators (CRISPR ON) to the proximal promoter of NOX1 and NOX2 and found reduced transcription of both genes in the presence of HDAC inhibitors. In the rat monocrotaline model of PH, we found that HDAC3‐5 were upregulated along with NOX2, while treatment with HDAC inhibitor Valproic acid reduced NOX2 expression and ameliorated PH. In conclusion NOX enzyme expression in multiple cell types is strongly influenced by epigenetic mechanisms. HDAC inhibitors, by reducing chromatin accessibility decrease NOX enzyme expression and ROS production which are likely to contribute to the anti‐inflammatory actions of these drugs and may, at least in part, explain their effectiveness in diseases such as PH.

Support or Funding Information

NIH, P01HL101902