Ji Qiang, Wang Yu Lin, Xia Li Min, Yang Ye, Wang Chun Sheng, Mei Yun Qing
Department of Cardiovascular Surgery of Zhongshan Hospital, Fudan University, 180 Fenglin Road, Shanghai, 200032, China.
Shanghai Institute of Cardiovascular Diseases, 1609 Xietu Rd, Shanghai, 200032, China.
J Cardiothorac Surg. 2019 Dec 12;14(1):216. doi: 10.1186/s13019-019-1025-5.
Early neointimal hyperplasia of vein graft may be ameliorated via enhancing intravenous surface shear stress. Cellular processes including proliferation, apoptosis and migration of endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) may play very important roles in the process of neointimal hyperplasia of vein graft; and mitogen-activated protein kinase (MAPK) pathways including extracellular signal-regulated kinase (ERK1/2) and p38 pathways play vital roles in regulating a large variety of cellular processes. This study evaluated the impacts of shear stress and MAPK pathways on cellular processes of ECs in a co-culture system with VSMCs, and aimed to test the hypothesis that high shear stress suppresses proliferation and migration but promotes apoptosis of ECs co-cultured with VSMCs via down-regulating MAPK pathway.
Primary ECs and VSMCs derived from porcine great saphenous vein were collected, respectively. 4-7 generation of cells were used as work cells. ECs and VSMCs were co-cultured and synchronized under high and low shear stress using Parallel-Plate Flow Chamber system. And then, ECs co-cultured with VSMCs were incubated with U0126 (ERK1/2 inhibitor) or PD98059 (p38 inhibitor) under different shear stress. Proliferation, apoptosis and migration of ECs in a co-culture system with VSMCs were detected by 4,5-dimethyl-2-thiazolyl (MTT) assay and bromodeoxyuridine (BrdU) assay, fluorescent-activated cell sorting (FACS) technique, and Transwell assay separately. Each test repeated 3 times. Additionally, protein expressions of ERK1/2 and p38 MAPK were detected by using Western blot, respectively.
Under higher level of shear stress condition, proliferation and migration of ECs co-cultured with VSMCs were suppressed, while cell apoptosis was promoted. And blocking ERK1/2 pathway by U0126 or blocking p38 pathway by PD98059, proliferation and migration of ECs co-cultured with VSMCs were further suppressed, while cell apoptosis was further promoted. Additionally, protein expressions of phosphorylation of ERK1/2 and p38MAPK were decreased under higher level of shear stress condition, and were further reduced by blocking ERK1/2 or p38 pathway under shear stress condition.
High shear stress may suppress proliferation and apoptosis of ECs in a co-culture system with VSMCs but promote cell migration via down-regulating ERK1/2 and p38 MAPK pathways.
通过增强静脉表面剪切应力可改善静脉移植物早期新生内膜增生。包括内皮细胞（ECs）和血管平滑肌细胞（VSMCs）的增殖、凋亡和迁移在内的细胞过程，在静脉移植物新生内膜增生过程中可能发挥非常重要的作用；而包括细胞外信号调节激酶（ERK1/2）和p38途径在内的丝裂原活化蛋白激酶（MAPK）途径，在调节多种细胞过程中起着至关重要的作用。本研究评估了剪切应力和MAPK途径对与VSMCs共培养体系中ECs细胞过程的影响，旨在验证高剪切应力通过下调MAPK途径抑制与VSMCs共培养的ECs的增殖和迁移，但促进其凋亡这一假说。
分别收集来自猪大隐静脉的原代ECs和VSMCs。使用第4 - 7代细胞作为工作细胞。利用平行板流动腔系统在高、低剪切应力下对ECs和VSMCs进行共培养并同步化。然后，在不同剪切应力下，将与VSMCs共培养的ECs与U0126（ERK1/2抑制剂）或PD98059（p38抑制剂）孵育。分别通过4,5 - 二甲基 - 2 - 噻唑基（MTT）法、溴脱氧尿苷（BrdU）法、荧光激活细胞分选（FACS）技术和Transwell法检测与VSMCs共培养体系中ECs的增殖、凋亡和迁移情况。每项检测重复3次。此外，分别使用蛋白质免疫印迹法检测ERK1/2和p38 MAPK的蛋白表达。
在较高水平的剪切应力条件下，与VSMCs共培养 的ECs的增殖和迁移受到抑制，而细胞凋亡得到促进。用U0126阻断ERK1/2途径或用PD98059阻断p38途径后，与VSMCs共培养的ECs的增殖和迁移进一步受到抑制，而细胞凋亡进一步得到促进。此外，在较高水平的剪切应力条件下，ERK1/2和p38MAPK的磷酸化蛋白表达降低，在剪切应力条件下阻断ERK1/2或p38途径后，其表达进一步降低。
高剪切应力可能通过下调ERK1/2和p38 MAPK途径抑制与VSMCs共培养体系中ECs的增殖和迁移，但促进细胞凋亡。
