Hong Zhongkui, Sun Zhe, Li Min, Li Zhaohui, Bunyak Filiz, Ersoy Ilker, Trzeciakowski Jerome P, Staiculescu Marius Catalin, Jin Minshan, Martinez-Lemus Luis, Hill Michael A, Palaniappan Kannappan, Meininger Gerald A
Dalton Cardiovascular Center, University of Missouri, 134 Research Park Dr., Columbia, MO 65211, USA.
J Physiol. 2014 Mar 15;592(6):1249-66. doi: 10.1113/jphysiol.2013.264929. Epub 2014 Jan 20.
In this study, we examined the ability of vasoactive agonists to induce dynamic changes in vascular smooth muscle cell (VSMC) elasticity and adhesion, and tested the hypothesis that these events are coordinated with rapid remodelling of the cortical cytoskeleton. Real-time measurement of cell elasticity was performed with atomic force microscopy (AFM) and adhesion was assessed with AFM probes coated with fibronectin (FN). Temporal data were analysed using an Eigen-decomposition method. Elasticity in VSMCs displayed temporal oscillations with three components at approximately 0.001, 0.004 and 0.07 Hz, respectively. Similarly, adhesion displayed a similar oscillatory pattern. Angiotensin II (ANG II, 10(-6) M) increased (+100%) the amplitude of the oscillations, whereas the vasodilator adenosine (ADO, 10(-4) M) reduced oscillation amplitude (-30%). To test whether the oscillatory changes were related to the architectural alterations in cortical cytoskeleton, the topography of the submembranous actin cytoskeleton (100-300 nm depth) was acquired with AFM. These data were analysed to compare cortical actin fibre distribution and orientation before and after treatment with vasoactive agonists. The results showed that ANG II increased the density of stress fibres by 23%, while ADO decreased the density of the stress fibres by 45%. AFM data were supported by Western blot and confocal microscopy. Collectively, these observations indicate that VSMC cytoskeletal structure and adhesion to the extracellular matrix are dynamically altered in response to agonist stimulation. Thus, vasoactive agonists probably invoke unique mechanisms that dynamically alter the behaviour and structure of both the VSMC cytoskeleton and focal adhesions to efficiently support the normal contractile behaviour of VSMCs.
在本研究中，我们检测了血管活性激动剂诱导血管平滑肌细胞（VSMC）弹性和黏附动态变化的能力，并验证了这些事件与皮质细胞骨架快速重塑相互协调的假说。使用原子力显微镜（AFM）对细胞弹性进行实时测量，并用包被纤连蛋白（FN）的AFM探针评估黏附情况。采用特征分解方法分析时间数据。VSMC中的弹性呈现出时间振荡，分别具有三个频率成分，约为0.001、0.004和0.07Hz。同样，黏附也呈现出类似的振荡模式。血管紧张素II（ANG II，10⁻⁶ M）使振荡幅度增加了100%，而血管舒张剂腺苷（ADO，10⁻⁴ M）使振荡幅度降低了30%。为了检测振荡变化是否与皮质细胞骨架的结构改变有关，用AFM获取了膜下肌动蛋白细胞骨架（深度100 - 300nm）的形貌。分析这些数据以比较血管活性激动剂处理前后皮质肌动蛋白纤维的分布和取向。结果显示，ANG II使应力纤维密度增加了2³%，而ADO使应力纤维密度降低了45%。AFM数据得到了蛋白质印迹和共聚焦显微镜的支持。总体而言，这些观察结果表明，VSMC细胞骨架结构和对细胞外基质的黏附会因激动剂刺激而发生动态改变。因此，血管活性激动剂可能引发独特的机制，动态改变VSMC细胞骨架和黏着斑的行为与结构，以有效支持VSMC的正常收缩行为。
