Wallis Lillian, Donovan Lucy, Johnston Aaron, Phillips Lauren C, Lin Jinheng, Garland Christopher J, Dora Kim A
The Vascular Pharmacology Group, Department of Pharmacology, University of Oxford, Oxford, United Kingdom.
Front Physiol. 2023 Jan 24;14:1108943. doi: 10.3389/fphys.2023.1108943. eCollection 2023.
Endothelial cell (EC) dysfunction is an early hallmark of cardiovascular disease associated with the reduced bioavailability of nitric oxide (NO) resulting in over-constriction of arteries. Despite the clear need to assess NO availability, current techniques do not reliably allow this in intact arteries. Confocal fluorescence microscopy was used to compare two NO-sensitive fluorescent dyes (NO-dyes), CuFL2E and DAR-4M AM, in both cell-free chambers and isolated, intact arteries. Intact rat mesenteric arteries were studied using pressure myography or imaging to visualize vascular smooth muscle cells (SMCs) and endothelial cells (ECs) under physiological conditions. Both NO-dyes irreversibly bind NO, so the time course of accumulated fluorescence during basal, EC-agonist (ACh, 1 µM), and NO donor (SNAP, 10 µM) responses were assessed and compared in all experimental conditions. To avoid motion artefact, we introduced the additional step of labelling the arterial elastin with AF-633 hydrazide (AF) and calculated the fluorescence ratio (FR) of NO-dye/elastin over time to provide data as FR/FR. In cell-free chambers using either CuFL2E or DAR-4M AM, the addition of SNAP caused a time-dependent and significant increase in fluorescence compared to baseline. Next, using pressure myography we demonstrate that both CuFL2E and DAR-4M AM could be loaded into arterial cells, but found each also labelled the elastin. However, despite the use of different approaches and the clear observation of NO-dye in SMCs or ECs, we were unable to measure increases in fluorescence in response to either ACh or SNAP when cells were loaded with CuFL2E. We then turned our attention to DAR-4M AM and observed increases in FR/FR following stimulation with either ACh or SNAP. The addition of each agent evoked an accumulating, time-dependent, and statistically significant increase in fluorescence within 30 min compared to time controls. These experiments were repeated in the presence of L-NAME, an NO synthase inhibitor, which blocked the increase in fluorescence on addition of ACh but not to SNAP. These data advance our understanding of vascular function and in the future will potentially allow us to establish whether ECs continuously release NO, even under basal conditions.
内皮细胞（EC）功能障碍是心血管疾病的早期标志，与一氧化氮（NO）生物利用度降低相关，导致动脉过度收缩。尽管明确需要评估NO的可用性，但目前的技术无法在完整动脉中可靠地进行此项评估。共聚焦荧光显微镜用于比较两种对NO敏感的荧光染料（NO染料），CuFL2E和DAR-4M AM，分别用于无细胞腔室和分离的完整动脉。使用压力肌动描记法或成像研究完整的大鼠肠系膜动脉，以在生理条件下可视化血管平滑肌细胞（SMC）和内皮细胞（EC）。两种NO染料都不可逆地结合NO，因此在所有实验条件下评估并比较了基础、EC激动剂（乙酰胆碱，1µM）和NO供体（硝普钠，10µM）反应期间累积荧光的时间进程。为避免运动伪影，我们引入了用AF-633酰肼（AF）标记动脉弹性蛋白的额外步骤，并计算随时间变化的NO染料/弹性蛋白荧光比率（FR），以提供FR/FR数据。在使用CuFL2E或DAR-4M AM的无细胞腔室中，添加硝普钠导致荧光与基线相比随时间呈依赖性显著增加。接下来，使用压力肌动描记法，我们证明CuFL2E和DAR-4M AM都可以加载到动脉细胞中，但发现每种染料也标记了弹性蛋白。然而，尽管使用了不同的方法，并且在SMC或EC中清楚地观察到了NO染料，但当细胞加载CuFL2E时，我们无法测量对乙酰胆碱或硝普钠的荧光增加。然后我们将注意力转向DAR-4M AM，并观察到用乙酰胆碱或硝普钠刺激后FR/FR增加。与时间对照相比，添加每种试剂在30分钟内引起荧光累积、时间依赖性且具有统计学意义的增加。在一氧化氮合酶抑制剂L-NAME存在的情况下重复这些实验，L-NAME可阻断添加乙酰胆碱时的荧光增加，但不阻断硝普钠引起的荧光增加。这些数据增进了我们对血管功能的理解，并且在未来可能使我们能够确定即使在基础条件下EC是否持续释放NO。
