Department of Surgery, Division of Cardiothoracic Surgery, University of Colorado-Denver, Aurora, Colorado 80045, USA.
J Surg Res. 2011 Mar;166(1):52-8. doi: 10.1016/j.jss.2009.03.101. Epub 2009 May 14.
Inflammation is involved in the pathogenesis of calcific aortic valve stenosis, and aortic valve interstitial cells (AVICs) play an important role in valve disease and remodeling. We have previously shown that human AVICs express functional toll-like receptor 4 (TLR4), and express increased levels of ICAM-1 in response to stimulation with bacterial lipopolysaccharide (LPS). This study examines (1) the role of TLR4 in regulating ICAM-1 expression in human AVICs, (2) the interaction of ICAM-1 with microfilaments, and (3) the influence of microfilament integrity on ICAM-1 expression.
AVICs isolated from explanted human hearts were stimulated with LPS (0.2 μg/mL) to induce ICAM-1 expression, as shown by Western blot. TLR4 activity was influenced with a neutralizing antibody or by gene knockdown with siRNA. Flow cytometry and immunofluorescence were used to characterize membrane localization of ICAM-1. Immunoprecipitation and immunofluorescence were employed to show colocalization of ICAM-1 with microfilaments. Microfilament function was influenced with an actin polymerization inhibitor as well as siRNA.
Cellular ICAM-1 levels increased 10-fold after 24 h of LPS stimulation, and this increase was significantly attenuated by prior treatment with TLR4-neutralizing antibody or TLR4 siRNA. ICAM-1 exhibited a filamentous distribution pattern after 4 h LPS stimulation, which was associated with increased total and cell-surface ICAM-1 expression. ICAM-1 colocalized and coprecipitated with the microfilament proteins F-actin and α-actinin 1. Pretreatment with the actin polymerization inhibitor latrunculin B and silencing α-actinin 1 with siRNA reduced LPS-induced total and cell-surface ICAM-1 expression.
This study demonstrates that (1) LPS-stimulated ICAM-1 expression in human AVICs is mediated by TLR4, (2) ICAM-1 interacts with microfilaments soon after LPS stimulation, and (3) microfilament disruption reduces ICAM-1 expression. These results suggest that the interaction between ICAM-1 and microfilaments facilitates LPS-induced ICAM-1 expression in human AVICs.
炎症参与了钙化性主动脉瓣狭窄的发病机制，而主动脉瓣间质细胞（AVICs）在瓣膜疾病和重塑中起着重要作用。我们之前已经证明，人 AVICs 表达功能性 Toll 样受体 4（TLR4），并在受到细菌脂多糖（LPS）刺激时表达增加的细胞间黏附分子 1（ICAM-1）。本研究探讨了（1）TLR4 在调节人 AVICs 中 ICAM-1 表达中的作用，（2）ICAM-1 与微丝的相互作用，以及（3）微丝完整性对 ICAM-1 表达的影响。
从心脏瓣膜中分离出的 AVICs 用 LPS（0.2μg/ml）刺激以诱导 ICAM-1 表达，Western blot 显示。TLR4 活性受中和抗体或 siRNA 基因敲低的影响。流式细胞术和免疫荧光用于表征 ICAM-1 的膜定位。免疫沉淀和免疫荧光用于显示 ICAM-1 与微丝的共定位。用肌动蛋白聚合抑制剂和 siRNA 影响微丝功能。
LPS 刺激 24 小时后，细胞内 ICAM-1 水平增加了 10 倍，而先用 TLR4 中和抗体或 TLR4 siRNA 预处理则显著减弱了这种增加。LPS 刺激 4 小时后，ICAM-1 呈现出丝状分布模式，与总 ICAM-1 和细胞表面 ICAM-1 表达增加有关。ICAM-1 与微丝蛋白 F-肌动蛋白和 α-辅肌动蛋白 1 共定位和共沉淀。用肌动蛋白聚合抑制剂 latrunculin B 预处理和用 siRNA 沉默 α-辅肌动蛋白 1 降低了 LPS 诱导的总 ICAM-1 和细胞表面 ICAM-1 表达。
本研究表明（1）LPS 刺激人 AVICs 中的 ICAM-1 表达是由 TLR4 介导的，（2）ICAM-1 在 LPS 刺激后立即与微丝相互作用，（3）微丝破坏减少了 ICAM-1 的表达。这些结果表明，ICAM-1 与微丝的相互作用促进了 LPS 诱导的人 AVICs 中 ICAM-1 的表达。
