Reducing Atherosclerotic Plaque Development and Endothelial Hemichannel Activity with WIN-55,212-2: A Research Protocol

Abstract

Introduction: Endothelial cells (ECs) are critical regulators of vascular homeostasis, and their dysfunction leads to the development of atherosclerosis – the main underlying cause of cardiovascular diseases (CVDs). This dysfunction can be promoted by prolonged endothelial connexin43 hemichannel activity, which is caused by the combination of high glucose levels and cytokines IL-1β/TNF-α. WIN-55,212-2 (WIN) is a synthetic agonist of CB1/CB2 receptors and can counteract the proinflammatory effects of high glucose and IL-1β/TNF-α. We hypothesize that WIN treatment on ECs will reduce connexin43 hemichannel activity, thus preventing endothelial dysfunction and atherosclerotic progression. Methods: We will use the Apolipoprotein E Knockout (ApoE-/-) mouse model to assess the impact on atherosclerotic lesions. Hyperglycemia will be generated in these mice with Streptozotocin injections. The increased levels of glucose should induce IL-1β expression and stimulate prolonged hemichannel activity. ECs will be isolated from a subset of mice and cultured to test WIN-efficacy. ATP release will be assessed through an ATP viability assay. More in vitro assessments on subsets of ApoE-/- mice treated or not with WIN will be performed. Flow cytometry will evaluate monocyte-derived macrophage concentration and other pro and anti-inflammatory cytokines in tissue samples. Furthermore, atherosclerotic plaque volume in the aortic sinus will be quantified and characterized. Results: We expect that WIN-treated ECs will reduce ATP synthesis compared to those from the control group. Moreover, we expect to see a reduction in the inflammatory response with a consequent decrease in atherosclerotic progression. Discussion: This manuscript outlines the use of a novel compound that could prevent atherosclerosis progression. The results of this study could outline a potential mechanism that may be targeted to treat or forestall atherosclerosis progression. Conclusion: Overall, we aim to determine if WIN may not only hinder this pervasive condition but inhibit CVDs through curtailing atherosclerotic plaque development. The following steps include performing the experiment, confirming results through repetition, and using other animal models.