Bartosch Anne Marie W, Mathews Rick, Tarbell John M
Department of Biomedical Engineering, The City College of New York, New York, New York.
Department of Biomedical Engineering, The City College of New York, New York, New York; The William E. Macaulay Honors College, New York, New York.
Biophys J. 2017 Jul 11;113(1):101-108. doi: 10.1016/j.bpj.2017.05.033.
Nitric oxide (NO) is a regulatory molecule in the vascular system and its inhibition due to endothelial injury contributes to cardiovascular disease. The glycocalyx is a thin layer of glycolipids, glycoproteins, and proteoglycans on the surface of mammalian epithelial cells. Extracellular forces are transmitted through the glycocalyx to initiate intracellular signaling pathways. In endothelial cells (ECs), previous studies have shown the glycocalyx to be a significant mediator of NO production; degradation of the endothelial glycocalyx layer (EGL) drastically reduces EC production of NO in response to fluid shear stress. However, the specific EGL components involved in this process are not well established. Recent work using short-hairpin RNA approaches in vitro suggest that the proteoglycan glypican-1, not syndecan-1, is the dominant core protein mediating shear-induced NO production. We utilized atomic force microscopy (AFM) to apply force selectively to components of the EGL of confluent rat fat pad ECs (RFPECs), including proteoglycans and glycosaminoglycans, to observe how each component individually contributes to force-induced production of NO. 4,5-diaminofluorescein diacetate, a cell-permeable fluorescent molecule, was used to detect changes in intracellular NO production. Antibody-coated AFM probes exhibited strong surface binding to RFPEC monolayers, with 100-300 pN mean adhesion forces. AFM pulling on glypican-1 and heparan sulfate for 10 min caused significantly increased NO production, whereas pulling on syndecan-1, CD44, hyaluronic acid, and with control probes did not. We conclude that AFM pulling can be used to activate EGL-mediated NO production and that the heparan sulfate proteoglycan glypican-1 is a primary mechanosensor for shear-induced NO production.
一氧化氮（NO）是血管系统中的一种调节分子，由于内皮损伤导致其抑制会引发心血管疾病。糖萼是哺乳动物上皮细胞表面的一层由糖脂、糖蛋白和蛋白聚糖组成的薄层。细胞外作用力通过糖萼传递，以启动细胞内信号通路。在内皮细胞（ECs）中，先前的研究表明糖萼是NO产生的重要介质；内皮糖萼层（EGL）的降解会显著降低ECs在流体剪切应力作用下产生NO的能力。然而，参与这一过程的特定EGL成分尚未完全明确。最近在体外使用短发夹RNA方法进行的研究表明，蛋白聚糖磷脂酰肌醇蛋白聚糖-1而非 syndecan-1是介导剪切诱导的NO产生的主要核心蛋白。我们利用原子力显微镜（AFM）选择性地对汇合的大鼠脂肪垫ECs（RFPECs）的EGL成分施加力，这些成分包括蛋白聚糖和糖胺聚糖，以观察每种成分如何单独促进力诱导的NO产生。4,5-二氨基荧光素二乙酸酯是一种可透过细胞的荧光分子，用于检测细胞内NO产生的变化。抗体包被的AFM探针与RFPEC单层表现出强烈的表面结合，平均粘附力为100 - 300 pN。对磷脂酰肌醇蛋白聚糖-1和硫酸乙酰肝素施加AFM拉力10分钟会导致NO产生显著增加，而对syndecan-1、CD44、透明质酸施加拉力以及使用对照探针则不会。我们得出结论，AFM拉力可用于激活EGL介导的NO产生，并且硫酸乙酰肝素蛋白聚糖磷脂酰肌醇蛋白聚糖-1是剪切诱导的NO产生的主要机械传感器。
