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Diabetes is a major health concern in the United States, with 1.5 million new cases diagnosed each year. Patients who suffer from diabetes have an increased risk of developing heart failure, a form of cardiovascular disease. Heart failure has been shown to result from increased left ventricular stiffness, which in turn is caused by increased remodeling of the extracellular matrix (ECM). This increase in ECM remodeling is a result of AGE/RAGE signaling, which occurs at a heightened level in the cardiac fibroblast cells of diabetics. Studies have shown that diabetics have elevated levels of AGEs (Advanced Glycation End-Products), which bind to RAGEs (Receptors for Advanced Glycation End-Products) on the cell surface, causing AGE/RAGE signaling to occur. AGE/RAGE signaling has also been demonstrated to play a role in oxidative stress. In addition to heart muscle stiffening, oxidative stress in the myocardium has also been found to correlate with left ventricular dysfunction. In this study we examined the impact of AGE/RAGE signaling on the relative expression levels of three different proteins associated with oxidative stress: p-NF-κB, SOD-1, and SOD-2. To accomplish this, we isolated cardiac fibroblasts from mice of six different genotypes: non-diabetic, diabetic, non-diabetic RAGE knockout, diabetic RAGE knockout, Rap1a wildtype, and Rap1a knockout. After isolation, we then grew those cells on plates that either had no underlying collagen (plastic), non-diabetic collagen (low endogenous AGEs), or diabetic collagen (elevated endogenous AGEs). We also treated some groups of fibroblasts with the pharmacological modifier EPAC, an allosteric activator of the enzyme Rap1a, to further alter the level of AGE/RAGE signaling. After treatment, proteins were isolated to v assess changes in the expression of oxidative stress markers via western blot analysis. Recently, research in our lab has identified that Rap1a, a small GTPase, may bisect the AGE/RAGE signaling pathway and contribute to the downstream outcomes. In light of this new data, we were also interested in assessing the impact of Rap1a on the AGE/RAGE-driven oxidative stress response. We found that increasing AGE/RAGE signaling led to an increase in expression of p-NF-κB and a decrease in expression of SOD-1 and SOD-2. In addition, we found that Rap1a appears to play an important role in the AGE/RAGE cascade by augmenting the downstream effects of this signaling pathway.
Dorroh, Christopher, "The Impact of AGE/RAGE Signaling on Oxidative Stress Under Diabetic Conditions in Cardiac Fibroblasts" (2020). Honors Theses. 1372.
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