舞蹈病蛋白对血管内皮细胞肌动蛋白聚合、细胞形状和机械硬度的敏感性

Chorein sensitivity of actin polymerization, cell shape and mechanical stiffness of vascular endothelial cells.

作者信息

Alesutan Ioana, Seifert Jan, Pakladok Tatsiana, Rheinlaender Johannes, Lebedeva Aleksandra, Towhid Syeda T, Stournaras Christos, Voelkl Jakob, Schäffer Tilman E, Lang Florian

机构信息

Department of Physiology, University of Tübingen, Tübingen, Germany.

出版信息

Cell Physiol Biochem. 2013;32(3):728-42. doi: 10.1159/000354475. Epub 2013 Sep 13.

DOI:10.1159/000354475

PMID:24080826

Abstract

BACKGROUND/AIMS: Endothelial cell stiffness plays a key role in endothelium-dependent control of vascular tone and arterial blood pressure. Actin polymerization and distribution of microfilaments is essential for mechanical cell stiffness. Chorein, a protein encoded by the VPS13A gene, defective in chorea-acanthocytosis (ChAc), is involved in neuronal cell survival as well as cortical actin polymerization of erythrocytes and blood platelets. Chorein is expressed in a wide variety of further cells, yet nothing is known about the impact of chorein on cells other than neurons, erythrocytes and platelets. The present study explored whether chorein is expressed in human umbilical vein endothelial cells (HUVECs) and addressed the putative role of chorein in the regulation of cytoskeletal architecture, stiffness and survival of those cells.

METHODS

In HUVECs with or without silencing of the VPS13A gene, VPS13A mRNA expression was determined utilizing quantitative RT-PCR, cytoskeletal organization visualized by confocal microscopy, G/F actin ratio and phosphorylation status of focal adhesion kinase quantified by western blotting, cell death determined by flow cytometry, mechanical properties studied by atomic force microscopy (AFM) and cell morphology analysed by scanning ion conductance microscopy (SICM).

RESULTS

VPS13A mRNA expression was detectable in HUVECs. Silencing of the VPS13A gene attenuated the filamentous actin network, decreased the ratio of soluble G-actin over filamentous F-actin, reduced cell stiffness and changed cell morphology as compared to HUVECs silenced with negative control siRNA. These effects were paralleled by a significant decrease in FAK phosphorylation following VPS13A silencing. Moreover, silencing of the VPS13A gene increased caspase 3 activity and induced necrosis in HUVECs.

CONCLUSIONS

Chorein is a novel regulator of cytoskeletal architecture, cell shape, mechanical stiffness and survival of vascular endothelial cells.

摘要

背景/目的：内皮细胞硬度在血管张力和动脉血压的内皮依赖性控制中起关键作用。肌动蛋白聚合和微丝分布对于细胞机械硬度至关重要。Chorein是一种由VPS13A基因编码的蛋白质，在舞蹈病-棘红细胞增多症（ChAc）中存在缺陷，它参与神经元细胞存活以及红细胞和血小板的皮质肌动蛋白聚合。Chorein在多种其他细胞中也有表达，但对于Chorein对神经元、红细胞和血小板以外的细胞的影响尚不清楚。本研究探讨了Chorein是否在人脐静脉内皮细胞（HUVECs）中表达，并研究了Chorein在调节这些细胞的细胞骨架结构、硬度和存活方面的假定作用。

方法

在VPS13A基因沉默或未沉默的HUVECs中，利用定量逆转录聚合酶链反应（RT-PCR）测定VPS13A mRNA表达，通过共聚焦显微镜观察细胞骨架组织，用蛋白质印迹法定量G/F肌动蛋白比率和粘着斑激酶的磷酸化状态，通过流式细胞术测定细胞死亡，用原子力显微镜（AFM）研究机械性能，并用扫描离子电导显微镜（SICM）分析细胞形态。

结果

在HUVECs中可检测到VPS13A mRNA表达。与用阴性对照小干扰RNA（siRNA）沉默的HUVECs相比，VPS13A基因沉默减弱了丝状肌动蛋白网络，降低了可溶性G-肌动蛋白与丝状F-肌动蛋白的比率，降低了细胞硬度并改变了细胞形态。这些效应与VPS13A沉默后粘着斑激酶磷酸化的显著降低并行。此外，VPS13A基因沉默增加了半胱天冬酶3活性并诱导了HUVECs坏死。