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LR-90 prevents methylglyoxal-induced oxidative stress and apoptosis in human endothelial cells

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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.

Conflict of interest

The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Departments of Diabetes and Metabolic Diseases Research, Beckman Research Institute of the City of Hope National Medical Center, NCI Designated Comprehensive Cancer Center, Gonda North, RM # 2108, 1500 E. Duarte Rd, Duarte, CA, 91010, USA

    James L. Figarola, Jyotsana Singhal, Samuel Rahbar, Sanjay Awasthi & Sharad S. Singhal

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  1. James L. Figarola
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  2. Jyotsana Singhal
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Correspondence to Sharad S. Singhal.

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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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