Transcriptional regulation of Hexokinase-2 by BRD4 drives perivascular adipose tissue meta-inflammation in cardiometabolic disease

Abstract

Aim: To investigate BRD4-related transcriptional programmes in mouse and human models of cardiometabolic disease. Methods: Small arteries (0.1-0.3 mm) dissected from visceral fat biopsies from healthy subjects (n=16) and patients with obesity and hypertension (n=16) were mounted on a pressurized myograph to assess the acute 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 by Western blot and qPCR. Transcriptional changes upon BRD4 inhibition were investigated by a custom PCR array, confirmed by ChIP, and characterised by metabolomics, lipidomics and mitochondrial swelling. Results: Endothelial-dependent vasorelaxation and vascular and perivascular TNF-alpha, IL-1beta, IL-6 were altered in cardiometabolic patients and mice. RVX-208 substantially attenuated ex-vivo vascular dysfunction, with an impact greater than 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 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 validated the findings by demonstrating a glycolytic shift in PVAT under disease conditions. Finally, ex vivo selective inhibition of HK2 rescued vascular dysfunction. Conclusion: Targeting the deleterious BRD4-HK2 interplay restores cardiometabolic vascular dysfunction via reversal of the PVAT meta-inflammatory shift, highlighting a novel potential target to fight cardiometabolic pandemics.