Cardioprotection by Metformin in Diabetic Mouse Coronary Artery Endothelial Cells Exposed to Hypoxia/Reoxygenation

Undergraduate #53
Discipline: Biological Sciences
Subcategory: Cell and Molecular Biology
Session: 1
Room: Exhibit Hall A

Jasmine S. Carter - Claflin University
Co-Author(s): Matthias L. Riess, Vanderbilt University, Nashville, TN; Michele M. Salzman, Vanderbilt University, Nashville, TN

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Type 2 diabetes mortality has increased significantly due to increased cardiovascular comorbidities. As a result, the development of an atherogenic profile occurs, in which blood accumulates free fatty acids known as hypertriglyceridemia. These have been shown to cause endothelial dysfunction due to a lack of nitric oxide, resulting in induced lipoapoptosis in endothelial cells (ECs). This can be detrimental since ECs serve a vital purpose to maintain blood vessel homeostasis by supporting vasodilation, fibrinolysis, and anti-aggregation to prevent atherosclerosis. Metformin, a first-line antidiabetic agent, has cardioprotective properties, in addition to lowering blood glucose levels. Metformin treatment protects against lipotoxicity of ECs by phosphorylation of endothelial nitric oxide synthase to increase nitric oxide production. The objective of the study was to determine the effect of metformin pretreatment on normal and high glucose diabetic mouse coronary artery ECs undergoing hypoxia/reoxygenation (H/R) injury in-vitro. We hypothesized that metformin attenuates the damage caused by high glucose and H/R injury. ECs were subjected to complete media with a glucose concentration of 5.5 mM (normal glucose) or 20.5 mM (high glucose), in addition to being subjected to metformin concentrations of 0 ?M, 30 ?M, 100 ?M, 300 ?M, or 1 mM for 24 or 72 hours for pretreatment. Following the metformin pretreatment, the ECs were exposed to normoxic (21% O2) or hypoxic (0.0125% O2) conditions for 8 hours and given a reoxygenation period of recovery for 16 hours. To assess the extent of injury and effect of metformin pretreatment, cell and mitochondrial viability, as well as cellular injury by lactate dehydrogenase (LDH) release, were measured. For the 24 and 72 hours of metformin pretreatment, ECs subjected to normal glucose had an approximately 40% hypoxic damage compared to ECs subjected to high glucose, which showed an approximately 30% hypoxic damage. Mitochondrial viability was greater after undergoing H/R for the ECs subjected to normal and high glucose for 24 and 72 hours of metformin pretreatment. In essence, for the 24 and 72 hours of metformin pretreatment, there was a higher level of LDH release for the ECs subjected to normal and high glucose undergoing H/R injury. Thus, ECs may not be a primary target for cardioprotection by metformin. Therefore, as a result of our current model, we plan to continue experiments with longer exposure periods of high glucose with a higher concentration than 20.5 mM. We also plan to perform metformin dose-dependent experiments to determine the concentrations that have a cardioprotective effect on the ECs.

Funder Acknowledgement(s): I would like to thank the National Heart, Lung, and Blood Institute and the American Heart Association for providing the funding for my project through the Promoting Academic Excellence with Community Engagement and Research Scholars Program (PAECER Program) (1R25HL145330). I also would like to thank the U.S. Department of Veterans Affairs Biomedical Laboratory R&D Service (Merit Review Award I01 BX003482 to Dr. Riess).

Faculty Advisor: Dr. Matthias L. Riess, matthias.riess@vanderbilt.edu

Role: I performed cell culture on diabetic mouse coronary endothelial cells. When the cells became confluent, I subjected them to normal (5.5 m) and high (20.5 mM) glucose concentrations and various metformin concentrations for pretreatment. Afterward, I exposed the cells to normoxic or hypoxic conditions for 8 hours and gave them a reoxygenation period for 16 hours. To determine the extent of damage and effect of the metformin pretreatment, I performed cell and mitochondrial viability assays, in addition to measuring the lactate dehydrogenase release for cellular injury. Following the collection of data, I entered the data into Excel and summarized the data into graphs and analyzed them.