Wang Huang-Joe, Lo Wan-Yu
Department of Internal Medicine, School of Medicine, China Medical University, Taichung, Taiwan.
Cardiovascular Research Laboratory, Division of Cardiovascular Medicine, Department of Internal Medicine, China Medical University and Hospital, Taichung, Taiwan.
Front Physiol. 2018 Jan 5;8:1095. doi: 10.3389/fphys.2017.01095. eCollection 2017.
Endothelial dysfunction is a hallmark of cardiovascular diseases. The straight region of an artery is protected from atherosclerosis via its laminar blood flow and high shear stress. This study investigated the cytoprotective effects of a new laminar shear medium (LSM) derived from a modified cone-and-plate shear device and identified basic fibroblast growth factor (bFGF) secreted by human aortic endothelial cells (HAECs) as the dominant protective factor in the LSM. Based on a modified cone-and-plate shear device system, HAECs were exposed to laminar shear (15 dynes/cm) and static control for 24 h to produce a new supernatant LSM and static medium (SM). Evaluation of the protective effects of LSM and SM on endothelial dysfunction induced by tumor necrosis factor (TNF)-α (10 ng/mL), which leads to production of reactive oxygen species (ROS), inflammatory monocyte adhesion, and tissue factor activity. ROS induction-, inflammation-, and thrombosis-related genes and protein expression were evaluated by quantitative-PCR and western blotting. To identify the cytokines that played a key role in the cytoprotective action of the LSM, we used cytokine antibody arrays, selected an abundant marker cytokine, bFGF, and validated the different cytoprotective effects of recombinant bFGF (rbFGF) and neutralization by monoclonal antibody (rbFGF+Ab) co-treatment. Aortic and lung tissues from different groups of C57BL/6J mice were examined by immunohistochemistry. SB203580 (specific inhibitor of p38) and BIX02189 (specific inhibitor of MEK5) were used to identify bFGF as the main cytoprotective factor acting via p38/MAPK and MEK5-KLF2 pathways. Compared with traditional LSM, the new LSM not only significantly decreased TNF-α-induced intracellular adhesion molecule 1 and plasminogen activator inhibitor type 1 gene expression, but also significantly increased heme oxygenase 1 gene expression. The new LSM and bFGF attenuated TNF-α-induced ROS induction, inflammation, and tissue factor activity and inhibited the inflammatory- and thrombosis-related gene/protein overexpression both and . Mechanistically, the cytoprotective action of bFGF was mediated via the p38/MAPK and MEK5-KLF2 pathways. bFGF was identified as the critical factor mediating the cytoprotective effects of LSM derived from the modified laminar shear system.
内皮功能障碍是心血管疾病的一个标志。动脉的直管区域通过其层流血液和高剪切应力来预防动脉粥样硬化。本研究调查了一种源自改良锥板剪切装置的新型层流剪切培养基（LSM）的细胞保护作用，并确定人主动脉内皮细胞（HAECs）分泌的碱性成纤维细胞生长因子（bFGF）是LSM中的主要保护因子。基于改良的锥板剪切装置系统，将HAECs暴露于层流剪切（15达因/平方厘米）和静态对照24小时，以产生新的上清液LSM和静态培养基（SM）。评估LSM和SM对肿瘤坏死因子（TNF）-α（10纳克/毫升）诱导的内皮功能障碍的保护作用，TNF-α会导致活性氧（ROS）产生、炎性单核细胞黏附以及组织因子活性。通过定量PCR和蛋白质印迹法评估ROS诱导、炎症和血栓形成相关基因及蛋白质表达。为了确定在LSM的细胞保护作用中起关键作用的细胞因子，我们使用细胞因子抗体阵列，选择了一种丰富的标记细胞因子bFGF，并通过重组bFGF（rbFGF）和单克隆抗体中和（rbFGF+Ab）联合处理验证了不同的细胞保护作用。通过免疫组织化学检查不同组C57BL/6J小鼠的主动脉和肺组织。使用SB203580（p38的特异性抑制剂）和BIX02189（MEK5的特异性抑制剂）来确定bFGF是通过p38/丝裂原活化蛋白激酶（MAPK）和MEK5-KLF2途径发挥作用的主要细胞保护因子。与传统LSM相比，新型LSM不仅显著降低了TNF-α诱导的细胞间黏附分子1和纤溶酶原激活物抑制剂1型基因表达，还显著增加了血红素加氧酶1基因表达。新型LSM和bFGF减轻了TNF-α诱导的ROS产生、炎症和组织因子活性，并抑制了炎症和血栓形成相关基因/蛋白质的过度表达。从机制上讲，bFGF的细胞保护作用是通过p38/MAPK和MEK5-KLF2途径介导的。bFGF被确定为介导源自改良层流剪切系统的LSM细胞保护作用的关键因子。
