Wei Lei, Ouyang Jing-Ping, Li Ke, Muller Sylvaine, Stoltz Jean-François, Wang Xiong
Department of Pathophysiology Medical College of Wuhan University, Wuhan 430071, China.
Zhongguo Ying Yong Sheng Li Xue Za Zhi. 2004 Nov;20(4):313-7.
To investigate the effects of different level of laminar shear stresses on the vasodilator-stimulated phosphoprotein (VASP) location and expression changes associated with actin reorganization and it's mechanism.
A parallel-plate flow chamber device was used to create laminar shear stress in vitro on cultured human umbilical endothelial cells (HUVECs). The distribution of VASP and microfilament were observed by double immunofluorescence staining. RT-PCR was used to test VASP mRNA level, while VASP parameters were analyzed quantitatively with Western blot.
After exposure to a flow of 10 dyn/cm2 flow for 24 h, HUVECs were elongated and oriented gradually to the direction of the flow. The microfilaments were recruited and oriented to the direction of flow with thicker VASP, specially targeted to the ends of stress fibres. RT-PCR result indicated shear could induce VASP mRNA increase. Western blotting data showed a dynamic reversible phosphorylation of VASP during 24 h, and total VASP expression increased rapidly, peaked at 2 h, then recovered at 8h followed by a slow increase again. H89, a cAMP inhibitor could inhibit shear induced VASP expression increase and phosphorylation.
VASP is an potential important component which participates in the regulation of cell cytoskeleton reorganization and morphology modification induced by shear flow via a cAMP/cAK pathway.
研究不同水平的层流切应力对血管舒张刺激磷蛋白（VASP）定位及表达变化的影响，及其与肌动蛋白重组的关系和机制。
采用平行平板流动腔装置在体外培养的人脐静脉内皮细胞（HUVECs）上施加层流切应力。通过双重免疫荧光染色观察VASP和微丝的分布。用RT-PCR检测VASP mRNA水平，同时用蛋白质免疫印迹法定量分析VASP参数。
在10达因/平方厘米的切应力作用24小时后，HUVECs逐渐伸长并沿血流方向排列。微丝被募集并沿血流方向排列，VASP变粗，特别定位于应力纤维末端。RT-PCR结果表明切应力可诱导VASP mRNA增加。蛋白质免疫印迹数据显示，在24小时内VASP发生动态可逆磷酸化，VASP总表达迅速增加，在2小时达到峰值，然后在8小时恢复，随后再次缓慢增加。cAMP抑制剂H89可抑制切应力诱导的VASP表达增加和磷酸化。
VASP是一个潜在的重要成分，它通过cAMP/cAK途径参与切变流诱导的细胞骨架重组和形态改变的调节。
