Synthetic 3D Model of the Carotid Artery to Study Exercise-Induced Changes in Endothelial Gene Expression

摘要 大约十分之九的脑血管疾病是由动脉粥样硬化引起的。此外，内皮细胞 功能障碍目前被认为是导致动脉粥样硬化的第一个病理生理步骤。内皮细胞 动态平衡和基因表达是通过切应力高度调节的，切应力与血液直接相关。 流量会发生变化。有氧运动(AX)与改善心血管(CV)健康有关。然而， 只有~50%的AX的有益影响是通过对传统的CV风险因素的改进来解释的(例如， 高血压、高胆固醇血症、肥胖)。AX剩余~50%的有益效果是 未知。此外，传统控制的AX不提供个性化药物，这可能会导致 对于大量无响应者。因此，这项建议的主要目的是开发一种3D 利用3D生物打印技术模拟活体模拟人颈动脉合成模型 个体化AX诱导的血流模式和血管内皮细胞切应力及基因测定 体外培养内皮细胞的表达/转录及分子变化基于以前的 报告和我们的初步数据假设，颈动脉的3D合成模型将对 运动诱导的血流模式与正常颈动脉相似。此外，我们假设内皮细胞 在相似的血流模式和切应力下培养的细胞会增加表达 保护动脉粥样硬化的信使核糖核酸/蛋白质(例如eNOS、PGI2和SOD)和结构信使核糖核酸/蛋白质(例如， 肌动蛋白、硫酸肝素蛋白多糖[糖萼]和α-肌动素束应激纤维)，以及前 动脉粥样硬化和促炎基因/蛋白(如ICAM-1、VCAM-1和ET-1)在类似的 强度依赖的方式。首先，我们将确定生物力学特性(例如，血管扩张性和 健康青年在体颈动脉在静息状态和3AX强度下的顺应性) 受试者、中风患者和年龄匹配的对照组。然后，受试者将接受磁共振检查 成像(MRI)研究，以确定相同测试颈动脉的确切形状(例如，长度和轮廓) 这些图像将被用来通过3D生物打印建立3D合成模型。3D合成模型将模拟 更多的解剖学和血流动力学条件，这将允许更多的生理体外实验。 其次，我们将在种植在3D合成模型上的内皮细胞中执行几种流动模式。 流动模式将类似于在活体研究中观察到的模式。在应用不同的流之后 模式，细胞将被收集和处理，以确定特定基因转录因子和 蛋白质表达。通过表征不同强度AX期间的血流模式，并确定 相同血流条件下内皮细胞的基因表达/转录及分子变化 模式，使用‘反向’翻译的方法，我们将解释心血管疾病的机制 与AX相关的保护性因素作为个性化药物，特别是在中风幸存者中。

Summary Approximately nine out of 10 cerebrovascular attacks are due to atherosclerosis. Additionally, endothelial dysfunction is currently accepted as the first pathophysiological step toward atherosclerosis. Endothelial cell homeostasis and gene expression is highly regulated via shear stress, which is directly associated with blood flow changes. Aerobic exercise (AX) has been associated with improved cardiovascular (CV) health. However, only ~50% of the beneficial effects of AX are explained via improvements on traditional CV risk factors (e.g., hypertension, hypercholesterolemia, obesity). The remainder ~50% of the beneficial effects of AX are unknown. Moreover, traditionally controlled AX does not provide personalized medicine, which could account for a high number of non-responders. Therefore, the main purpose of this proposal is to develop a 3D synthetic model of the human carotid artery using 3D bio-printing technology to simulate in vivo personalized AX-induced blood flow patterns and endothelial shear stress and to determine gene expression/transcription and molecular changes in endothelial cultured cells in vitro. Based on previous reports and our preliminary data we hypothesize that a 3D synthetic model of the carotid artery will respond to exercise-induced blood flow patterns as a normal carotid artery. In addition, we hypothesize that endothelial cultured cells under similar blood flow patterns and shear stress will increase the expression of atherosclerosis-protective mRNA/proteins (e.g., eNOS, PGI2, and SOD) and structural mRNA/proteins (e.g., actin, heparin sulfate proteoglycan [glycocalyx], and α-actinin-bundled stress fibers), and a decrease of pro- atherosclerosis and pro-inflammatory mRNA/protein expression (e.g., ICAM-1, VCAM-1, and ET-1) in a similar intensity-dependent manner. First, we will determine biomechanical properties (e.g., vessel distensibility and compliance) of the carotid artery in vivo during resting conditions and at 3 AX intensities in healthy, young subjects, patients with stroke, and age-matched controls. Then, subjects will undergo a magnetic resonance imaging (MRI) study to determine the exact shape (e.g., length and contour) of same tested carotid artery and the images will be used to build a 3D synthetic model via 3D bio-printing. The 3D synthetic model will mimic more anatomical and hemodynamic conditions, which will allow for more physiological in vitro experiments. Secondly, we will perform several flow patterns in endothelial cultured cells seeded on the 3D synthetic model. Flow patterns will be similar to those patterns observed during in vivo studies. After applying the different flow patterns, cells will be collected and processed to determine changes in specific gene transcription factors and protein expression. By characterizing blood flow patterns during different intensities of AX and determining the gene expression/transcription and molecular changes in endothelial cells under these same blood flow patterns, using a ‘reverse’ translation approach, we will explain the mechanisms of the cardiovascular protective factors associated with AX as personalized medicine, especially in stroke survivors.