Neurovascular Center, Changhai Hospital, Naval Medical University, No. 168 Changhai Rd, Shanghai, 200433, China.
Department of Neurosurgery, General Hospital of Northern Theater Command, 83 Wenhua Road, Shenyang, Liaoning Province, China.
BMC Neurosci. 2022 Nov 5;23(1):61. doi: 10.1186/s12868-022-00748-2.
In brain, microvascular endothelial cells are exposed to various forces, including shear stress (SS). However, little is known about the effects of high shear stress (HSS) on human brain microvascular endothelial cells (HBMECs) and the underlying mechanism. The cholesterol efflux regulator ATP-binding cassette subfamily A member 1 (ABCA1) has been demonstrated to exert protective effect on HBMECs. However, whether ABCA1 is involved in the mechanism underneath the effect of HSS on HBMECs remains obscure. In the present study, a series of experiments were performed to better understand the effect of HSS on cellular processes of HBMECs and the possible involvement of ABCA1 and PI3K/Akt/eNOS in the underlying mechanisms.
HBMECs were subjected to physiological SS (PSS) or high SS (HSS). Cell migration was evaluated using Transwell assay. Apoptotic HBMECs were detected by flow cytometry or caspase3/7 activity. IL-1β, IL-6, MCP-1 and TNF-α levels were measured by ELISA. RT-qPCR and western blotting were used for mRNA and protein expression detection, respectively. ROS and NO levels were detected using specific detection kits. Compared to PSS, HBMECs exhibited decreased cell viability and migration and increased cell apoptosis, increased levels of inflammatory cytokines, and improved ROS and NO productions after HSS treatment. Moreover, HSS downregulated ABCA1 but upregulated the cholesterol efflux-related proteins MMP9, AQP4, and CYP46 and activated PI3K/Akt/eNOS pathway. Overexpression of ABCA1 in HBMECS inhibited PI3K/Akt/eNOS pathway and counteracted the deleterious effects of HSS. Contrary effects were observed by ABCA1 silencing. Inhibiting PI3K/Akt/eNOS pathway mimicked ABCA1 effects, suggesting that ABCA1 protects HBMECs from HSS via PI3K/Akt/eNOS signaling.
These results advanced our understanding on the mechanisms of HSS on HBMECs and potentiated ABCA1/PI3K/Akt/eNOS pathway as therapeutic target for cerebrovascular diseases.
在大脑中，微血管内皮细胞会受到各种力的作用，包括切应力（SS）。然而，目前对于高切应力（HSS）对人脑微血管内皮细胞（HBMEC）的影响及其潜在机制知之甚少。胆固醇外排调节剂 ATP 结合盒亚家族 A 成员 1（ABCA1）已被证明对 HBMEC 具有保护作用。然而，ABCA1 是否参与 HSS 对 HBMEC 作用的机制尚不清楚。在本研究中，进行了一系列实验，以更好地了解 HSS 对 HBMEC 细胞过程的影响，以及 ABCA1 和 PI3K/Akt/eNOS 是否参与潜在机制。
将 HBMEC 暴露于生理 SS（PSS）或高 SS（HSS）中。通过 Transwell 测定评估细胞迁移。通过流式细胞术或 caspase3/7 活性检测凋亡的 HBMEC。通过 ELISA 测定 IL-1β、IL-6、MCP-1 和 TNF-α 水平。使用 RT-qPCR 和 Western blotting 分别用于检测 mRNA 和蛋白表达。使用特定的检测试剂盒检测 ROS 和 NO 水平。与 PSS 相比，HSS 处理后 HBMEC 的细胞活力和迁移降低，细胞凋亡增加，炎症细胞因子水平升高，ROS 和 NO 生成增加。此外，HSS 下调 ABCA1，但上调胆固醇外排相关蛋白 MMP9、AQP4 和 CYP46，并激活 PI3K/Akt/eNOS 途径。ABCA1 在 HBMEC 中的过表达抑制了 PI3K/Akt/eNOS 途径，并抵消了 HSS 的有害影响。ABCA1 沉默则观察到相反的效果。抑制 PI3K/Akt/eNOS 途径模拟了 ABCA1 的作用，表明 ABCA1 通过 PI3K/Akt/eNOS 信号通路保护 HBMEC 免受 HSS 的影响。
这些结果加深了我们对 HSS 对 HBMEC 的影响机制的理解，并增强了 ABCA1/PI3K/Akt/eNOS 途径作为脑血管疾病的治疗靶点。
