BHF Centre of Research Excellence, Division of Cardiovascular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford OX3 9DU, UK.
Alzheimer's Research UK, Oxford Drug Discovery Institute, Target Discovery Institute, Nuffield Department of Medicine, University of Oxford, Oxford OX3 7FZ, UK.
Int J Mol Sci. 2022 Apr 28;23(9):4891. doi: 10.3390/ijms23094891.
Angiogenesis is a critical cellular process toward establishing a functional circulatory system capable of delivering oxygen and nutrients to the tissue in demand. In vitro angiogenesis assays represent an important tool for elucidating the biology of blood vessel formation and for drug discovery applications. Herein, we developed a novel, high content 2D angiogenesis assay that captures endothelial morphogenesis's cellular processes, including lumen formation. In this assay, endothelial cells form luminized vascular-like structures in 48 h. The assay was validated for its specificity and performance. Using the optimized assay, we conducted a phenotypic screen of a library containing 150 FDA-approved cardiovascular drugs to identify modulators of lumen formation. The screening resulted in several L-type calcium channel blockers being able to expand the lumen space compared to controls. Among these blockers, Lacidipine was selected for follow-up studies. We found that the endothelial cells treated with Lacidipine showed enhanced activity of caspase-3 in the luminal space. Pharmacological inhibition of caspase activity abolished the Lacidipine-enhancing effect on lumen formation, suggesting the involvement of apoptosis. Using a Ca biosensor, we found that Lacipidine reduces the intracellular Ca oscillations amplitude in the endothelial cells at the early stage, whereas Lacidipine blocks these Ca oscillations completely at the late stage. The inhibition of MLCK exhibits a phenotype of lumen expansion similar to that of Lacidipine. In conclusion, this study describes a novel high-throughput phenotypic assay to study angiogenesis. Our findings suggest that calcium signalling plays an essential role during lumen morphogenesis. L-type Ca channel blockers could be used for more efficient angiogenesis-mediated therapies.
血管生成是建立能够向需求组织输送氧气和营养物质的功能性循环系统的关键细胞过程。体外血管生成测定法是阐明血管形成生物学和药物发现应用的重要工具。在此，我们开发了一种新颖的高通量 2D 血管生成测定法，该方法可以捕获内皮细胞形态发生的细胞过程，包括管腔形成。在该测定法中，内皮细胞在 48 小时内形成有管腔的血管样结构。该测定法的特异性和性能已得到验证。使用优化的测定法，我们对包含 150 种 FDA 批准的心血管药物的文库进行了表型筛选，以鉴定管腔形成的调节剂。筛选结果表明，几种 L 型钙通道阻滞剂能够与对照相比扩大管腔空间。在这些阻滞剂中，拉西地平被选中进行后续研究。我们发现，用拉西地平处理的内皮细胞在管腔空间中 caspase-3 的活性增强。 caspase 活性的药理学抑制消除了拉西地平对管腔形成的增强作用，表明细胞凋亡的参与。使用 Ca 生物传感器，我们发现拉西地平在早期阶段降低内皮细胞中细胞内 Ca 振荡的幅度，而拉西地平在晚期阶段完全阻断这些 Ca 振荡。 MLCK 的抑制作用表现出与拉西地平相似的管腔扩张表型。总之，这项研究描述了一种用于研究血管生成的新型高通量表型测定法。我们的研究结果表明，钙信号在管腔形态发生过程中起着至关重要的作用。 L 型钙通道阻滞剂可用于更有效的血管生成介导的治疗。
