Targeting BRD4-HK2 reverses the meta-inflammatory shift in perivascular adipose tissue and rescues cardiometabolic vascular dysfunction

Abstract Background/Introduction Reducing vascular inflammation is crucial to halt and reverse cardiometabolic damage. BRD4, an epigenetic pan-inflammatory activator whose inhibition has shown promising results in cancer, may play an important role in this context. However, its role in cardiometabolic disease remains unclear. Purpose To investigate BRD4-related transcriptional programmes and therapeutic modulation in translational models of cardiometabolic disease. Methods We dissected small arteries (100-300 μM) from visceral fat biopsies in healthy volunteers (n=16) and patients with obesity and hypertension (n=16), a highly prevalent cardiometabolic phenotype. We assessed the ex vivo effects of BRD4 inhibition on vascular function by pressure myography in the presence or absence of perivascular adipose tissue (PVAT), at baseline and after incubation with the BRD4 inhibitor RVX-208 or with anti-inflammatory/anti-metabolic drugs. We used a cardiometabolic mouse model (high-fat diet+L-NAME supplementation) that mirrored our human phenotype and orally administered RVX-208 (150 mg/kg) or vehicle for 5 days (n=6 per group) to confirm the in vivo effect of chronic BRD4 inhibition. Finally, we investigated the molecular pathways involved in the perivascular cross-talk in an in vitro model of human adipocytes (hADSCs) and endothelial cells (HAECs) using gene overexpression/silencing strategies. We assessed ROS and nitric oxide levels (confocal microscopy), protein and gene expressions (Western blot; qPCR), transcriptional (custom qPCR array; chromatin immunoprecipitation (ChIP)) and metabolic changes (metabolomics, lipidomics, mitochondrial swelling). Results Vascular inflammation, remodeling and function were altered in cardiometabolic patients/mice. RVX-208 attenuated ex vivo vascular dysfunction to a greater extent than anti-IL-1β, anti-IL-6 receptor and anti-TNF-α. In vessels with intact PVAT we observed a stronger effect, due to the restoration of healthy PVAT phenotype (Figure 1). In PVAT, Hexokinase-2 (Hk2) - a glycolytic enzyme implicated in mitochondrial dysfunction and inflammation - was the top downregulated gene by RVX-208 treatment; ChIP confirmed increased binding of BRD4 to the Hk2 promoter. In line, metabolomics and lipidomics assays revealed a PVAT glycolytic shift with increased fatty acid storage in disease states, which was reversed by BRD4 inhibition. The in vitro study showed that: i) healthy adipocytes overexpressing HK2 shift to an inflam