Succinate Regulates Endothelial Mitochondrial Function and Barrier Integrity
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 resp...
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2024-12-01
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| Series: | Antioxidants |
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| author | Reham Atallah Juergen Gindlhuber Wolfgang Platzer Rishi Rajesh Akos Heinemann |
| author_facet | Reham Atallah Juergen Gindlhuber Wolfgang Platzer Rishi Rajesh Akos Heinemann |
| author_sort | Reham Atallah |
| collection | DOAJ |
| description | 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<sub>2</sub>/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. |
| format | Article |
| id | doaj-art-1b851c7514b640fc986d8287ccac8aab |
| institution | DOAJ |
| issn | 2076-3921 |
| language | English |
| publishDate | 2024-12-01 |
| publisher | MDPI AG |
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| series | Antioxidants |
| spelling | doaj-art-1b851c7514b640fc986d8287ccac8aab2025-08-20T02:57:08ZengMDPI AGAntioxidants2076-39212024-12-011312157910.3390/antiox13121579Succinate Regulates Endothelial Mitochondrial Function and Barrier IntegrityReham Atallah0Juergen Gindlhuber1Wolfgang Platzer2Rishi Rajesh3Akos Heinemann4Otto Loewi Research Center for Vascular Biology, Immunology and Inflammation, Division of Pharmacology, Medical University of Graz, 8010 Graz, AustriaOtto Loewi Research Center for Vascular Biology, Immunology and Inflammation, Division of Physiology & Pathophysiology, Medical University of Graz, 8010 Graz, AustriaOtto Loewi Research Center for Vascular Biology, Immunology and Inflammation, Division of Pharmacology, Medical University of Graz, 8010 Graz, AustriaOtto Loewi Research Center for Vascular Biology, Immunology and Inflammation, Division of Pharmacology, Medical University of Graz, 8010 Graz, AustriaOtto Loewi Research Center for Vascular Biology, Immunology and Inflammation, Division of Pharmacology, Medical University of Graz, 8010 Graz, AustriaEndothelial 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<sub>2</sub>/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.https://www.mdpi.com/2076-3921/13/12/1579endothelial cellssuccinatemitochondrial functionbarrier integrityreactive oxygen species |
| spellingShingle | Reham Atallah Juergen Gindlhuber Wolfgang Platzer Rishi Rajesh Akos Heinemann Succinate Regulates Endothelial Mitochondrial Function and Barrier Integrity Antioxidants endothelial cells succinate mitochondrial function barrier integrity reactive oxygen species |
| title | Succinate Regulates Endothelial Mitochondrial Function and Barrier Integrity |
| title_full | Succinate Regulates Endothelial Mitochondrial Function and Barrier Integrity |
| title_fullStr | Succinate Regulates Endothelial Mitochondrial Function and Barrier Integrity |
| title_full_unstemmed | Succinate Regulates Endothelial Mitochondrial Function and Barrier Integrity |
| title_short | Succinate Regulates Endothelial Mitochondrial Function and Barrier Integrity |
| title_sort | succinate regulates endothelial mitochondrial function and barrier integrity |
| topic | endothelial cells succinate mitochondrial function barrier integrity reactive oxygen species |
| url | https://www.mdpi.com/2076-3921/13/12/1579 |
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