Atallah Reham, Gindlhuber Juergen, Platzer Wolfgang, Rajesh Rishi, Heinemann Akos
Otto Loewi Research Center for Vascular Biology, Immunology and Inflammation, Division of Pharmacology, Medical University of Graz, 8010 Graz, Austria.
Otto Loewi Research Center for Vascular Biology, Immunology and Inflammation, Division of Physiology & Pathophysiology, Medical University of Graz, 8010 Graz, Austria.
Antioxidants (Basel). 2024 Dec 21;13(12):1579. doi: 10.3390/antiox13121579.
Endothelial dysfunction is a hallmark of several pathological conditions, including cancer, cardiovascular disease and inflammatory disorders. In these conditions, perturbed TCA cycle and subsequent succinate accumulation have been reported. The role of succinate as a regulator of immunological responses and inflammation is increasingly being recognized. Nevertheless, how endothelial cell function and phenotype are altered by elevated intracellular succinate has not been addressed yet. Thus, we employed numerous in vitro functional assays using primary HUVECs and diethyl succinate (DES), a cell membrane-permeable succinate analogue. An MTS assay 1 h post stimulation with DES suggested reduced metabolic activity in HUVECs. Concurrently, elevated production of ROS, including mitochondrial superoxide, and a reduction in mitochondrial membrane potential were observed. These findings were corroborated by Seahorse mito-stress testing, which revealed that DES acutely lowered the OCR, maximal respiration and ATP production. Given the link between mitochondrial stress and apoptosis, we examined important survival signalling pathways. DES transiently reduced ERK1/2 phosphorylation, a response that was followed by a skewed pro-apoptotic shift in the BAX to BCL2L1 gene expression ratio, which coincided with upregulating VEGF gene expression. This indicated an induction of mixed pro-apoptotic and pro-survival signals in the cell. However, the BAX/BCL-XL protein ratio was unchanged, suggesting that the cells did not commit themselves to apoptosis. An MTS assay, caspase 3/7 activity assay and annexin V/propidium iodide staining confirmed this finding. By contrast, stimulation with DES induced acute endothelial barrier permeability, forming intercellular gaps, altering cell size and associated actin filaments without affecting cell count. Notably, during overnight DES exposure gradual recovery of the endothelial barrier and cell sprouting was observed, alongside mitochondrial membrane potential restoration, albeit with sustained ROS production. COX-2 inhibition and EP4 receptor blockade hindered barrier restoration, implicating a role of COX-2/PGE/EP4 signalling in this process. Interestingly, ascorbic acid pre-treatment prevented DES-induced acute barrier disruption independently from ROS modulation. In conclusion, succinate acts as a significant regulator of endothelial mitochondrial function and barrier integrity, a response that is counterbalanced by upregulated VEGF and prostaglandin production by the endothelial cells.
内皮功能障碍是包括癌症、心血管疾病和炎症性疾病在内的多种病理状况的一个标志。在这些病症中，已有报道三羧酸循环紊乱及随后的琥珀酸积累。琥珀酸作为免疫反应和炎症调节因子的作用日益受到认可。然而，细胞内琥珀酸水平升高如何改变内皮细胞功能和表型尚未得到研究。因此，我们使用原代人脐静脉内皮细胞（HUVECs）和琥珀酸二乙酯（DES，一种可透过细胞膜的琥珀酸类似物）进行了多项体外功能试验。DES刺激1小时后的MTS试验表明HUVECs的代谢活性降低。同时，观察到包括线粒体超氧化物在内的活性氧生成增加以及线粒体膜电位降低。海马体线粒体应激试验证实了这些发现，该试验表明DES可急性降低氧耗率、最大呼吸和ATP生成。鉴于线粒体应激与细胞凋亡之间的联系，我们研究了重要的生存信号通路。DES短暂降低了ERK1/2磷酸化，随后BAX与BCL2L1基因表达比值出现促凋亡倾向的变化，这与VEGF基因表达上调同时发生。这表明细胞内诱导了混合的促凋亡和促生存信号。然而，BAX/BCL-XL蛋白比值未变，表明细胞未进入凋亡程序。MTS试验、caspase 3/7活性试验和膜联蛋白V/碘化丙啶染色证实了这一发现。相比之下，DES刺激可诱导急性内皮屏障通透性增加，形成细胞间间隙，改变细胞大小和相关肌动蛋白丝，但不影响细胞数量。值得注意的是，在DES过夜暴露期间，观察到内皮屏障逐渐恢复和细胞发芽，同时线粒体膜电位恢复，尽管活性氧持续产生。COX-2抑制和EP4受体阻断阻碍了屏障恢复，表明COX-2/PGE/EP4信号在此过程中起作用。有趣的是，抗坏血酸预处理可独立于活性氧调节预防DES诱导的急性屏障破坏。总之，琥珀酸是内皮线粒体功能和屏障完整性的重要调节因子，内皮细胞上调VEGF和前列腺素生成可对这一反应起到平衡作用。
