Background Pulmonary arterial hypertension (PAH) is an incurable disease characterised by raised pulmonary resistance, resulting from vascular remodelling which leads to right heart failure and death. Recently NF-kB mediated inflammatory gene expression and vascular proliferation/remodelling have been shown to be involved in the pathogenesis of PAH. The expression of subsets of NF-kB-induced inflammatory genes is dependent upon the recruitment of the bromodomains and extra-terminal (BET) family of proteins to the transcriptional activation complex. We hypothesise that inhibition of BET proteins which bind acetylated lysine residues on histones and non-histone proteins will attenuate the hyperproliferative and proinflammatory phenotype of vascular cells.
Methods Primary human pulmonary vascular endothelial cells (P-EC) were serum starved for 24 h prior to treatment with the Brd4 mimic JQ1+ or JQ1- (inactive enantiomer) in complete (5% FCS) media. P-EC cell proliferation was measured by BrdU incorporation and apoptosis was determined using caspase 3/7 activity. Cell cycle progression was determined by FACs analysis. mRNA levels of cell cycle genes and inflammatory cytokines were measured by RT-PCR. MTT assay was used to measure cell viability.
Results JQ1+ caused a significant (p < 0.001) and concentration-dependent decrease in P-EC proliferation and an increase in caspase 3/7 activity compared to P-EC treated with JQ1- for 24 h. JQ1+ (1 µM) significantly arrested the cell cycle of P-EC at the G1 phase. This was additionally evidenced by a decrease in the cell cycle genes CDK2, 4 and 6 mRNA levels and a significant increase in the mRNA of the cell cycle inhibitor CDKN1A (p21/cip1) at 4 h. Finally, JQ1+ significantly (p < 0.01) inhibited the mRNA levels of the inflammatory cytokines IL-6 and 8 in P-EC compared to JQ1–.
Conclusion Inhibition of Brd4 with JQ1 decreases remodelling and inflammation in P-EC via a decrease in proliferation, cell cycle arrest and an increase in apoptosis. Further work is required but Brd4 inhibition may provide therapeutic drugs for the treatment of PAH.