A Bio-Engineering Approach to Endothelial Cell Signal Transduction and Responses to Fluid Shear Stress

我们在一个特别设计的流加载室中对培养的内皮细胞施加可控水平的流体剪切应力，并检测细胞内Ca^<2+>浓度（[Ca^<2+>]）和粘附分子表达的变化。当细胞外ATP存在的情况下受到剪切应力时，ECs显示出[Ca^<2+>]的增加。在单个细胞中观察到不同的模式，包括峰值和平台，振荡和瞬态。该模式的比例分别为62%、32%和6%。无论模式如何，[Ca^<2+>]增加从细胞边缘的特定位点开始，并以Ca^<2+>波的形式在整个细胞体中传播。这些位点与ECs中富含小泡蛋白的位点相对应，表明剪切应力信息是通过小泡进入的。剪切应力抑制小鼠淋巴结小静脉内皮细胞表面血管粘附分子-1 （vascular adhesion molecule-1, VCAM-1）的表达，同时降低其mRNA水平。mRNA水平的下降是由于VCAM-1基因转录受到抑制，而VCAM-1启动子中的双AP1共识元件对于剪切诱导的转录活性抑制至关重要。这是负剪应力响应元件，这是本研究首次证明的。

We applied controlled levels of fluid shear stress to cultured endothelial cells (ECs) in a specially designed flow-loading chamber, and examined the changes in intracellular Ca^<2+> concentrations ([Ca^<2+>]) and adhesion molecule expressions.When subjected to shear stress in the presence of extracellular ATP,ECs showed the increase in [Ca^<2+>]. Different patterns including peak & plateau, oscillation, and transient were observed in individual cells. The percentage of the pattern was 62%, 32%and6%, respectively. Regardless of the patterns, [Ca^<2+>] increase initiated from specific loci at cell edges, and propagated through the entire cell body as a Ca^<2+> wave. The loci corresponded to the caveolin-rich in ECs, suggesting that the information of shear stress enters via caveolae.Shear stress inhibited the cell surface expression of vascular adhesion molecule-1 (VCAM-1) in mouse lymphnode venule ECs, and simultaneously decreased the mRNA levels. The decrease in mRNA levels was due to the suppression of VCAM-1 gene transcription, and double AP1 consensus elements in VCAM-1 promoter was essential for the shear-induced suppression of transcriptional activity. This is a negative shear stress responsive element, which was first demonstrated by this study.