Differential responses of pulmonary vascular cells from PAH patients and controls to TNFα and the effect of the BET inhibitor JQ1

BackgroundPulmonary arterial hypertension (PAH) encompasses a group of diseases characterized by raised pulmonary vascular resistance, resulting from vascular remodelling and inflammation. Bromodomain and extra-terminal (BET) proteins are required for the expression of a subset of NF-& kappa;B-induced inflammatory genes which can be inhibited by the BET mimic JQ1+. We hypothesised that JQ+ would supress TNF & alpha;-driven inflammatory responses in human pulmonary vascular cells from PAH patients.MethodsImmunohistochemical staining of human peripheral lung tissue (N = 14 PAH and N = 12 non-PAH) was performed for the BET proteins BRD2 and 4. Human pulmonary microvascular endothelial cells (HPMEC) and pulmonary artery smooth muscle cells (HPASMC) from PAH patients (N = 4) and non-PAH controls (N = 4) were stimulated with TNF & alpha; in presence or absence of JQ1+ or its inactive isomer JQ1-. IL-6 and -8 mRNA was measured by RT-qPCR and protein levels by ELISA. Chromatin immunoprecipitation analysis was performed using EZ-ChIP & TRADE; and NF-& kappa;B p65 activation determined using a TransAm kit. MTT assay was used to measure cell viability.ResultsNuclear staining of BRD2 and BRD4 was significantly (p < 0.0001) increased in the lung vascular endothelial and smooth muscle cells from PAH patients compared to controls with normal lung function. TNF & alpha;-driven IL-6 release from both HPMECs and HPASMCs was greater in PAH cells than control cells. Levels of CXCL8/IL-8 protein release was higher in PAH HPASMCs than in control cells with similar release observed in HPMECs. TNF & alpha;-induced recruitment of activated NF-& kappa;B p65 to the IL-6 and CXCL8/IL-8 promoters were similar in both cell types and between subject groups. JQ1+ suppressed TNF & alpha;-induced IL-6 and CXCL8/IL-8 release and mRNA expression to a comparable extent in control and PAH HPMECs and HPASMCs. JQ1 had a greater efficacy on IL-6 release in HPMEC and on CXCL8/IL-8 release in HPASMC.ConclusionBET inhibition decreases TNF & alpha; driven inflammation in primary pulmonary vascular cells. The anti-inflammatory actions of JQ1 suggests distinct cell-specific regulatory control of these genes. BET proteins could be a target for future therapies for PAH.