Wei Lei, Muller Sylvaine, Ouyang JingPing, Stoltz Jean-François, Wang Xiong
Group Mechanics and Cell and Tissue Engineering, LEMTA-UMR 7563 CNRS/INPL/UHP and IFR 111 Bioengineering, 54500 Vandoeuvre-lès-Nancy, France.
Clin Hemorheol Microcirc. 2003;28(2):113-20.
Vasodilator-stimulated phosphoprotein (VASP), an actin filaments-associated protein expressed mainly in focal adhesions and dynamic membrane regions of endothelial cells (ECs), serves as a substrate for cAMP and cGMP-dependent protein kinases. In this work, we studied the effect of laminar shear stress in vitro on the location and expression of the VASP as well as its phosphorylation associated with actin reorganisation in human umbilical endothelial cells (HUVECs). The distributions of VASP and microfilaments were observed by a fluorescent double staining. The level of VASP expression in cells was quantified by western blot. Experiments showed that, after exposure to a shear stress of 10 dyn/cm(2) for 24 h, besides the elongation and orientation of the cells, and spots of VASP were found along thick stress fibres, particularly at their two extremities. Western blot data showed conversions up to 11.4% and 8.9% of the 46 kD non-phosphorylated form VASP to its 50 kD phosphorylated form after exposures of 1 h and 4 h respectively to the same shear stress. The shear stress of 15 dyn/cm(2) had qualitatively the same effect as that of 10 dyn/cm(2), but with a lower magnitude. However, the shear stress of 2 dyn/cm(2) had much slower and weaker effects on the same tendency. These results suggest that a laminar shear stress can induce VASP translocation and phosphorylation that proceed actin filaments rearrangement along with the flow direction in HUVECs.
血管舒张刺激磷蛋白（VASP）是一种主要在内皮细胞（ECs）的粘着斑和动态膜区域表达的肌动蛋白丝相关蛋白，可作为环磷酸腺苷（cAMP）和环磷酸鸟苷（cGMP）依赖性蛋白激酶的底物。在本研究中，我们在体外研究了层流切应力对人脐静脉内皮细胞（HUVECs）中VASP的定位和表达及其与肌动蛋白重组相关的磷酸化的影响。通过荧光双重染色观察VASP和微丝的分布。通过蛋白质印迹法定量细胞中VASP的表达水平。实验表明，在暴露于10达因/平方厘米的切应力24小时后，除了细胞伸长和定向外，还发现VASP斑点沿着粗大的应力纤维分布，特别是在其两端。蛋白质印迹数据显示，在分别暴露于相同切应力1小时和4小时后，46kD非磷酸化形式的VASP分别有高达11.4%和8.9%转化为其50kD磷酸化形式。15达因/平方厘米的切应力在性质上与10达因/平方厘米的切应力相同，但程度较低。然而，2达因/平方厘米的切应力对相同趋势的影响要慢得多且弱得多。这些结果表明，层流切应力可诱导HUVECs中VASP易位和磷酸化，进而使肌动蛋白丝沿血流方向重排。
