Respiratory Science, NHLI, Imperial College London, London, UK.
Inserm UMR-S 999, Hôpital Marie Lannelongue, Groupe Hospitalier Paris Saint Joseph, Université Paris-Saclay, Le Plessis-Robinson, France.
Respir Res. 2023 Jul 29;24(1):193. doi: 10.1186/s12931-023-02499-y.
Pulmonary 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-κB-induced inflammatory genes which can be inhibited by the BET mimic JQ1+. We hypothesised that JQ+ would supress TNFα-driven inflammatory responses in human pulmonary vascular cells from PAH patients.
Immunohistochemical 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 TNFα 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™ and NF-κB p65 activation determined using a TransAm kit. MTT assay was used to measure cell viability.
Nuclear 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. TNFα-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. TNFα-induced recruitment of activated NF-κB p65 to the IL-6 and CXCL8/IL-8 promoters were similar in both cell types and between subject groups. JQ1+ suppressed TNFα-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.
BET inhibition decreases TNFα 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.
肺动脉高压（PAH）是一组以肺血管阻力升高为特征的疾病，其由血管重塑和炎症引起。Bromodomain 和 extra-terminal（BET）蛋白是 NF-κB 诱导的炎症基因表达所必需的，这些基因可以被 BET 模拟物 JQ1+抑制。我们假设 JQ+会抑制来自 PAH 患者的人肺血管细胞中 TNFα 驱动的炎症反应。
对 14 例 PAH 患者和 12 例非 PAH 患者的人外周肺组织进行 BET 蛋白 BRD2 和 4 的免疫组织化学染色。来自 PAH 患者（N=4）和非 PAH 对照（N=4）的人肺微血管内皮细胞（HPMEC）和肺动脉平滑肌细胞（HPASMC）在 TNFα 的存在或不存在下用 JQ1+或其无活性异构体 JQ1-刺激。通过 RT-qPCR 测量 IL-6 和 -8 mRNA 的水平，并通过 ELISA 测量蛋白水平。使用 EZ-ChIP™进行染色质免疫沉淀分析，并使用 TransAm 试剂盒测定 NF-κB p65 的激活。MTT 测定法用于测量细胞活力。
与具有正常肺功能的对照相比，PAH 患者的肺血管内皮和平滑肌细胞中 BRD2 和 BRD4 的核染色显著增加（p<0.0001）。来自 PAH 细胞的 TNFα 驱动的 HPMEC 和 HPASMC 中的 IL-6 释放比对照细胞更大。PAH HPASMC 中的 CXCL8/IL-8 蛋白释放高于对照细胞，而在 HPMEC 中观察到相似的释放。在两种细胞类型和两组受试者中，TNFα 诱导的活化 NF-κB p65 募集到 IL-6 和 CXCL8/IL-8 启动子的情况相似。在对照和 PAH HPMEC 和 HPASMC 中，JQ1+以相似的程度抑制了 TNFα 诱导的 IL-6 和 CXCL8/IL-8 释放和 mRNA 表达。JQ1 在 HPMEC 中对 IL-6 释放和在 HPASMC 中对 CXCL8/IL-8 释放的作用更大。
BET 抑制可减少原代肺血管细胞中 TNFα 驱动的炎症。JQ1 的抗炎作用表明这些基因存在独特的细胞特异性调控。BET 蛋白可能成为 PAH 未来治疗的靶点。
