Abstract
Methylglyoxal (MGO) is a highly reactive dicarbonyl compound known to induce cellular injury and cytoxicity, including apoptosis in vascular cells. Vascular endothelial cell apoptosis has been implicated in the pathophysiology and progression of atherosclerosis. We investigated whether the advanced glycation end-product inhibitor LR-90 could prevent MGO-induced apoptosis in human umbilical vascular endothelial cells (HUVECs). HUVECs were pre-treated with LR-90 and then stimulated with MGO. Cell morphology, cytotoxicity and apoptosis were evaluated by light microscopy, MTT assay, and Annexin V-FITC and propidium iodide double staining, respectively. Levels of Bax, Bcl-2, cytochrome c, mitogen-activated protein kinases (MAPKs) and caspase activities were assessed by Western blotting. Reactive oxygen species (ROS) generation and mitochondrial membrane potential (MMP) were measured with fluorescent probes. LR-90 dose-dependently prevented MGO-associated HUVEC cytotoxicity and apoptotic biochemical changes such as loss of MMP, increased Bax/Bcl-2 protein ratio, mitochondrial cytochrome c release and activation of caspase-3 and 9. Additionally, LR-90 blocked intracellular ROS formation and MAPK (p44/p42, p38, JNK) activation, though the latter seem to be not directly involved in MGO-induced HUVEC apoptosis. LR-90 prevents MGO-induced HUVEC apoptosis by inhibiting ROS and associated mitochondrial-dependent apoptotic signaling cascades, suggesting that LR-90 possess cytoprotective ability which could be beneficial in prevention of diabetic related-atherosclerosis.
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Abbreviations
- AG:
-
Aminoguanidine
- AGE:
-
Advanced glycation end-product
- ECs:
-
Endothelial cells
- HUVECs:
-
Human umbilical vascular endothelial cells
- LR-90:
-
Methylene bis [4,4′-(2 chlorophenylureido phenoxyisobutyric acid)]
- MAPKs:
-
Mitogen-activated protein kinases
- MGO:
-
Methylglyoxal
- MMP:
-
Mitochondrial membrane potential
- NAC:
-
N-acetyl cysteine
- ROS:
-
Reactive oxygen species
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Acknowledgments
This work was supported by the National Institutes of Health Grant (CA 77495). The authors are grateful to Jacquelin and Isaac Moradi for their many years of support and research funding. Funding from Department’s Chair (Prof. Arthur Riggs) and Beckman Research Institute of the City of Hope is also acknowledged. We are also thankful to Dr. Brian Armstrong (Microscope Core, City of Hope) and Lucy Brown (Analytical Core, City of Hope) for their technical assistance in microscopy and flow cytometry analyses, respectively.
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The authors declare that they have no conflict of interest.
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Figarola, J.L., Singhal, J., Rahbar, S. et al. LR-90 prevents methylglyoxal-induced oxidative stress and apoptosis in human endothelial cells. Apoptosis 19, 776–788 (2014). https://doi.org/10.1007/s10495-014-0974-3
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DOI: https://doi.org/10.1007/s10495-014-0974-3