Transcriptional Regulation of Hexokinase-2 by Brd4 Drives Perivascular Adipose Tissue Meta-Inflammation in Cardiometabolic Disease

Objective: To investigate BRD4-related transcriptional programmes and therapeutic modulation in mouse and human models of cardiometabolic disease. Design and method: Small arteries (0.1-0.3 mm) from healthy subjects (n=16) and patients with obesity and hypertension (n=16) were dissected from visceral fat biopsies and mounted on a pressurised myograph to assess the ex-vivo effects of BRD4 inhibition on vascular function. Vasorelaxation to acetylcholine and acetylcholine+L-NAME was evaluated, in the presence or in the absence of perivascular adipose tissue (PVAT), at baseline and after incubation with the BRD4 inhibitor RVX-208 and with selective anti-inflammatory and anti-metabolic drugs. A cardiometabolic mouse (high-fat diet+L-NAME supplementation) was orally administered RVX-208 (150 mg/kg) to test in vivo effect of chronic BRD4 inhibition. ROS and nitric oxide were assessed by confocal microscopy; protein and gene expression were measured by Western blot and qPCR. Transcriptional changes upon BRD4 inhibition were investigated by a custom qPCR array, confirmed by ChIP, qPCR and Western blot and further characterised by metabolomics, lipidomics and mitochondrial swelling assays. Results: Endothelial-dependent vasorelaxation and tissue levels of TNF-alpha, IL-1beta, and IL-6 were altered in the vessel wall and PVAT from cardiometabolic patients and mice. RVX-208 substantially attenuated ex-vivo vascular dysfunction, with an impact greater when compared to anti-IL-1beta, anti-IL-6 receptor and anti-TNF-alpha. The effect was more pronounced in vessels with intact PVAT, suggesting a restoration of the PVAT anti-contractile phenotype. Gene expression profiling in PVAT from cardiometabolic mice unveiled hexokinase-2 (HK2) - a glycolytic enzyme implicated in mitochondrial dysfunction and inflammation - as the top downregulated gene by RVX-208 treatment. Increased binding of BRD4 to HK2 promoter in PVAT samples from cardiometabolic mice was confirmed by ChIP assays. Metabolomics assays further validate the findings, showing a PVAT glycolytic shift in condition of disease. Finally, ex-vivo selective inhibition of HK2 restored vascular dysfunction. Conclusions: Targeting the deleterious BRD4-HK2 interplay restores cardiometabolic-related vascular dysfunction reversing the PVAT meta-inflammatory shift. Epigenetic modulators of meta-inflammatory pathways might represent a promising strategy to prevent and reverse vascular dysfunction, key signature of cardiometabolic disease.