Date of Award
James A Stewart, Jr.
T. Kristopher Harrell
Cardiovascular disease is the leading cause of death in the United States with one of the major contributors being vascular calcification, characterized by the deposition of calcium-phosphate hydroxyapatite. Once thought to be a passive process due to aging, medial vascular calcification is becoming better understood as a tightly regulated, cellmediated process. Vascular smooth muscle cells (VSMCs) within the medial arterial layer respond to inflammation, oxidative stress, and changes in pyrophosphate levels, which induce differentiation into osteoblast-like cells. Fibroblasts within the adventitial layer (AFBs) also alter their function in response to vascular calcification signaling, although this is largely understudied. The AGE/RAGE signaling cascade has been implicated in medial vascular calcification. We hypothesize that the small GTPase, Rap1a, contributes to AGE/RAGE signaling, resulting in vascular calcification. We chose to investigate Rap1a in order to better elucidate the molecular mechanisms behind the development of medial vascular calcification. In order to do this, in vitro studies were conducted using VSMCs and AFBs isolated from wild-type (WT) and Rap1a knockout (KO) mice to further investigate the possible role of Rap1a in vascular calcification. Cells were exposed to 3 mM inorganic phosphate to simulate physiological calcification conditions. Following seven days of treatment, a colorimetric calcification assay was utilized to quantify intracellular calcium levels. Expression of proteins such as RAGE, αsmooth muscle actin (αSMA), osteopontin (OPN), and toll-like receptor 4 (TLR-4) were v quantified via western blotting. WT VSMCs presented with equal amounts of calcification compared to KO except with the addition of AGEs in KO cells, suggesting that the role of Rap1a in the context of the AGE/RAGE cascade requires further investigation. However, our data suggests that WT and KO AFB calcification is dependent on the activation of the AGE/RAGE cascade. Therefore, we conclude that AFBs and VSMCs differ in their mechanisms of calcification formation and that further research is necessary to fully understand the role of AGE/RAGE and Rap1a in medial arterial vascular calcification.
Haydel, Amelia Grace, "Investigating the Role of Rap1a in Medial Arterial Vascular Calcification" (2020). Honors Theses. 1510.
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