内皮功能障碍,在心血管疾病(CVD)中普遍存在,并与糖尿病等疾病有关,高血压,肥胖,肾功能衰竭,或高胆固醇血症,其特征在于一氧化氮(NO)生物利用度降低-血管稳态的关键信号分子。目前关于内皮细胞(ECs)NO合成的二维(2D)体外研究缺乏关键的层流剪切应力,调节NO生成酶的一个重要因素,内皮型一氧化氮合酶(eNOS),在生理条件下。在这里,我们开发了一种基于示踪剂的代谢组学方法,通过质谱(MS)测量NO特异性代谢物,并使用2D和3D平台显示流体流动对与NO合成相关的代谢参数的影响。具体来说,我们追踪了稳定同位素标记的NO底物L-精氨酸向L-瓜氨酸和L-鸟氨酸的转化,以确定eNOS活性。我们证明了用13C6,15N4-L-精氨酸培养的人冠状动脉内皮细胞(HCAECs)的反应明显,用eNOS刺激器治疗,eNOS抑制剂,和精氨酸酶抑制剂。下游代谢物的分析,13C6,15N3L-瓜氨酸和13C5,15N2L-鸟氨酸,揭示了不同的结果。此外,我们评估了具有双向和单向流体流动的静态2D培养和3D微血管模型中的NO代谢状态。我们的3D模型表现出显著的效果,特别是在暴露于eNOS刺激器的微血管中,如13C6、15N3L-瓜氨酸/13C5、15N2L-鸟氨酸比率所示,与2D文化相比。获得的结果表明,2D静态培养模拟内皮功能障碍状态,而具有单向流体流动的3D模型提供了更具代表性的生理环境,为研究内皮功能障碍提供了更好的模型。
Endothelial dysfunction, prevalent in cardiovascular diseases (CVDs) and linked to conditions like diabetes, hypertension, obesity, renal failure, or hypercholesterolemia, is characterized by diminished nitric oxide (NO) bioavailability-a key signaling molecule for vascular homeostasis. Current two-dimensional (2D) in vitro studies on NO synthesis by endothelial cells (ECs) lack the crucial laminar shear stress, a vital factor in modulating the NO-generating enzyme, endothelial nitric oxide synthase (eNOS), under physiological conditions. Here we developed a tracer-based metabolomics approach to measure NO-specific metabolites with mass spectrometry (MS) and show the impact of fluid flow on metabolic parameters associated with NO synthesis using 2D and 3D platforms. Specifically, we tracked the conversion of stable-isotope labeled NO substrate L-Arginine to L-
Citrulline and L-Ornithine to determine eNOS activity. We demonstrated clear responses in human coronary artery endothelial cells (HCAECs) cultured with 13C6, 15N4-L-Arginine, and treated with eNOS stimulator, eNOS inhibitor, and arginase inhibitor. Analysis of downstream metabolites, 13C6, 15N3 L-
Citrulline and 13C5, 15N2 L-Ornithine, revealed distinct outcomes. Additionally, we evaluated the NO metabolic status in static 2D culture and 3D microvessel models with bidirectional and unidirectional fluid flow. Our 3D model exhibited significant effects, particularly in microvessels exposed to the eNOS stimulator, as indicated by the 13C6, 15N3 L-
Citrulline/13C5, 15N2 L-Ornithine ratio, compared to the 2D culture. The obtained results indicate that the 2D static culture mimics an endothelial dysfunction status, while the 3D model with a unidirectional fluid flow provides a more representative physiological environment that provides a better model to study endothelial dysfunction.