Date of Award
Biomedical Sciences (M.H.S.)
Atherosclerosis is the leading cause of cardiovascular diseases like myocardial infarctions and strokes. The formation of these atherosclerotic plaques occurs through localized inflammatory responses in blood vessels. Epidemiological evidence suggests omega-3 fatty acids (FAs) decrease vascular inflammation. The omega-3 FAs eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) reduce inflammation through pathways that may include the free fatty acid-4 (FFA-4) receptor. While EPA and DHA are well studied, less is known about the essential omega-3 FA from which they are synthesized: alpha-linolenic acid (ALA). Thus, this study will investigate whether ALA can activate the FFA-4 receptor and thereby contribute to anti-inflammatory responses in the endothelium. Although the FFA-4 receptor is a G-protein coupled receptor, its activation reduces the nuclear transcription factor NF-κB thereby decreasing the synthesis and release of inflammatory cytokines. The resulting decrease in inflammatory cytokines is expected to decrease expression of connexin-43 (Cx43). Cx43 is a gap junction protein whose endothelial cell expression is increased during inflammation contributing to an unhealthy endothelium. Treatment of endothelial cells with 30 M ALA is hypothesized to decrease the expression of Cx43 through the activation of the FFA-4 receptor. Cultured mouse endothelial cells (bEnd.3) were treated with physiological ALA concentrations (30 M) for 48 hours. Whole-cell protein was isolated from treated and controlled endothelial cells at 1.5, 3, 6, 12, 24 and 48 hours and expression of Cx43 determined. Over the 48 hours of ALA exposure, Cx43 expression did not change (ratio of Cx43 expression in ALA treated/time-control was not different from 1: p>0.05). Increasing ALA to 100 M did nothing to affect Cx43 expression in cells at 12 – 48 hours (p>0.05). While this suggests ALA is not involved in omega-3 FA dependent anti-inflammatory responses in the endothelium, these results could be affected by ALA activating the pro-inflammatory FFA-1 receptor. However, treatment of cells with 10 M GW-9508 (selective FFA-1 receptor agonist) did not affect Cx43 expression at 48 hours (p>0.05). Thus, treatment of bEnd.3 cells with ALA did not alter expression of Cx43. These results suggest ALA may not contribute to the anti-inflammatory influence omega-3 FAs exert on endothelial cells.
Pichette, Benjamin, "The effect of alpha-linolenic acid (ALA) on endothelial cell expression of connexin43" (2018). Masters Theses. 889.