Khalaf Maha, Scott-Ward Toby, Causer Adam, Saynor Zoe, Shepherd Anthony, Górecki Dariusz, Lewis Anthony, Laight David, Shute Janis
School of Pharmacy and Biomedical Sciences, Institute of Biological and Biomedical Sciences, University of Portsmouth, Portsmouth, United Kingdom.
Department of Sport and Exercise Science, University of Portsmouth, Portsmouth, United Kingdom.
Front Physiol. 2020 Jul 29;11:879. doi: 10.3389/fphys.2020.00879. eCollection 2020.
Perturbation of endothelial function in people with cystic fibrosis (CF) has been reported, which may be associated with endothelial cell expression of the cystic fibrosis transmembrane conductance regulator (CFTR). Previous reports indicate that CFTR activity upregulates endothelial barrier function, endothelial nitric oxide synthase (eNOS) expression and NO release, while limiting interleukin-8 (IL-8) release, in human umbilical vein endothelial cells (HUVECs) in cell culture. In view of reported microvascular dysfunction in people with CF we investigated the role of CFTR expression and activity in the regulation of oxidative stress, cell signaling and inflammation in human lung microvascular endothelial cells (HLMVECs) in cell culture.
HLMVECs were cultured in the absence and presence of the CFTR inhibitor GlyH-101 and CFTR siRNA. CFTR expression was analyzed using qRT-PCR, immunocytochemistry (IHC) and western blot, and function by membrane potential assay. IL-8 expression was analyzed using qRT-PCR and ELISA. Nrf2 expression, and NF-κB and AP-1 activation were determined using IHC and western blot. The role of the epidermal growth factor receptor (EGFR) in CFTR signaling was investigated using the EGFR tyrosine kinase inhibitor AG1478. Oxidative stress was measured as intracellular ROS and hydrogen peroxide (HO) concentration. VEGF and SOD-2 were measured in culture supernatants by ELISA.
HLMVECs express low levels of CFTR that increase following inhibition of CFTR activity. Inhibition of CFTR, significantly increased intracellular ROS and HO levels over 30 min and significantly decreased Nrf2 expression by 70% while increasing SOD-2 expression over 24 h. CFTR siRNA significantly increased constitutive expression of IL-8 by HLMVECs. CFTR inhibition activated the AP-1 pathway and increased IL-8 expression, without effect on NF-κB activity. Conversely, TNF-α activated the NF-κB pathway and increased IL-8 expression. The effects of TNF-α and GlyH-101 on IL-8 expression were additive and inhibited by AG1478. Inhibition of both CFTR and EGFR in HLMVECs significantly increased VEGF expression. The antioxidant N-acetyl cysteine significantly reduced ROS production and the increase in IL-8 and VEGF expression following CFTR inhibition.
Functional endothelial CFTR limits oxidative stress and contributes to the normal anti-inflammatory state of HLMVECs. Therapeutic strategies to restore endothelial CFTR function in CF are warranted.
据报道，囊性纤维化（CF）患者存在内皮功能紊乱，这可能与囊性纤维化跨膜传导调节因子（CFTR）在内皮细胞中的表达有关。既往报道表明，在细胞培养的人脐静脉内皮细胞（HUVECs）中，CFTR活性上调可增强内皮屏障功能、内皮型一氧化氮合酶（eNOS）表达及一氧化氮（NO）释放，同时限制白细胞介素-8（IL-8）释放。鉴于CF患者存在微血管功能障碍的报道，我们研究了CFTR表达及活性在细胞培养的人肺微血管内皮细胞（HLMVECs）氧化应激、细胞信号传导及炎症调节中的作用。
在有无CFTR抑制剂GlyH-101及CFTR小干扰RNA（siRNA）的情况下培养HLMVECs。采用定量逆转录聚合酶链反应（qRT-PCR）、免疫细胞化学（IHC）及蛋白质印迹法分析CFTR表达，并通过膜电位测定分析其功能。采用qRT-PCR及酶联免疫吸附测定（ELISA）分析IL-8表达。采用IHC及蛋白质印迹法测定核因子E2相关因子2（Nrf2）表达以及核因子κB（NF-κB）和激活蛋白-1（AP-1）的激活情况。使用表皮生长因子受体（EGFR）酪氨酸激酶抑制剂AG1478研究EGFR在CFTR信号传导中的作用。以细胞内活性氧（ROS）及过氧化氢（HO）浓度衡量氧化应激。通过ELISA检测培养上清液中的血管内皮生长因子（VEGF）及超氧化物歧化酶2（SOD-2）。
HLMVECs表达低水平的CFTR，在CFTR活性受到抑制后其表达增加。抑制CFTR后，30分钟内细胞内ROS及HO水平显著升高，Nrf2表达显著降低70%，而24小时内SOD-2表达增加。CFTR siRNA显著增加HLMVECs组成性IL-8表达。抑制CFTR可激活AP-1通路并增加IL-8表达，但对NF-κB活性无影响。相反，肿瘤坏死因子-α（TNF-α）激活NF-κB通路并增加IL-8表达。TNF-α和GlyH-101对IL-8表达的影响具有相加性，且被AG1478抑制。抑制HLMVECs中的CFTR和EGFR可显著增加VEGF表达。抗氧化剂N-乙酰半胱氨酸可显著减少ROS生成以及CFTR抑制后IL-8和VEGF表达的增加。
功能性内皮CFTR可限制氧化应激，并有助于HLMVECs维持正常的抗炎状态。有必要采取治疗策略恢复CF患者内皮CFTR功能。
