Live imaging of nitric oxide release in vascular endothelial cells in response to mechanical stimuli on an organ chip
Live imaging of nitric oxide release in vascular endothelial cells in response to mechanical stimuli on an organ chip
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血管内皮细胞响应器官芯片上的机械刺激而释放一氧化氮的实时成像
DOI：
10.1093/eurheartj/ehac544.3027
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发表时间：
2022
影响因子：
39.3
通讯作者：
Naruse K
中科院分区：
文献类型：
--
作者：
Takahashi K;Liu Y;Wang M;Liang Y;Naruse K
血管内皮细胞对机械刺激的反应释放出一氧化氮(Nitric Oxide，NO)，调节心脏收缩能力，参与心脏肥大的预防。目的建立一种用于实时观察器官芯片对机械刺激的NO释放的实验系统。方法使用我们在先前研究中用于开发心脏芯片的器官芯片[1]。通过增加连接到芯片通道的蠕动泵的流量来对电池施加剪切力(图1A)。压力刺激由静水压力施加。拉伸刺激是通过使用电动注射器泵吸入芯片的侧端口来施加的(图1B)。用10μM4，5-二氨基荧光素二乙酸酯对细胞进行荧光染色，不显影。用德克萨斯红染料(MW3000)测得的表观通透性，一直维持在较低的∼3×10~(-6)cm/S水平，直到第30天，这表明细胞间连接的形成。当内皮细胞受到60 MmHg60 S的压力刺激时，没有观察到持续2分钟的释放(图2A)。在压力刺激开始(n=251个细胞)后，观察到峰值为基线的1.46±1.08(均值±标准差)倍于S。当细胞受到1%的拉伸时，60 S在拉伸刺激开始后观察到峰值为基线的1.29±0.33倍，105 S(图2B)。0.01dyn/cm2的切应力几乎不会增加NO的释放(1.20±0.27倍于基线，图2C)。结论利用芯片上器官建立了血管内皮细胞对机械刺激的活体NO成像系统。含有内皮细胞的心脏芯片将有助于阐明高血压等机械刺激对心脏收缩功能和心脏重塑的影响。基金确认资金来源类型：公共拨款(S)-仅国家预算。主要资金来源(S)：日本科学促进会图1图2
BackgroundNitric oxide (NO), released from vascular endothelial cells in response to mechanical stimuli, regulates cardiac contractility and are also involved in the prevention of the development of cardiac hypertrophy.PurposeTo establish an experimental system for live observation of NO release in response to mechanical stimuli on an organ chip.MethodsOrgan chips, which we used for the development of a heart-on-a-chip in the previous study [1], were used.We seeded 300,000 human umbilical vein endothelial cells on a stretchable elastic membrane coated with Matrigel of a chip channel. Shear stress was applied to the cells by increasing flow rate of a peristaltic pump connected to the chip channel (Figure 1A). Pressure stimulus was applied by hydrostatic pressure. Stretch stimulus was applied by suction to the side ports of a chip using an electric syringe pump (Figure 1B). Cells were stained with 10 μM 4,5-diaminofluorescein diacetate for fluorescent live NO imaging.ResultsMonolayers of the endothelial cells formed intercellular junctions confirmed by CD31 staining (Figure 1C, yellow). Apparent permeability, which was measured by Texas red dye (MW 3000), was maintained at a low level of ∼3x10–6cm/s until day 30, suggested the formation of robust intercellular junction.When the endothelial cells were subjected to a pressure stimulus of 60 mmHg for 60 s, NO release was observed that lasted for >2 minutes (Figure 2A). A peak value of 1.46±1.08 (mean ± standard deviation) times the baseline was observed 271 s after the beginning of the pressure stimulus (n=251 cells). When the cells were subjected to a 1% stretch for 60 s, a peak value of 1.29±0.33 times the baseline was observed 105 s after the beginning of the stretch stimulus (Figure 2B). A shear stress of 0.01 dyn/cm2hardly increased NO release (1.20±0.27 times the baseline, Figure 2C).ConclusionThe system for live NO imaging in vascular endothelial cells in response to mechanical stimuli was established using organ-on-a-chip. The heart-on-a-chip with endothelial cells will be useful in elucidating the effects of mechanical stimulus such as hypertension on the contractile function and the remodeling of the heart.Funding AcknowledgementType of funding sources: Public grant(s) – National budget only. Main funding source(s): Japan Society for the Promotion of ScienceFigure 1Figure 2