Abstract 354: Haploinsufficiency Of Sam68 Attenuates Vascular Inflammation And Atherosclerosis In Apoe-deficient Mice

Abstract

Background: Chronic inflammation in the arterial wall is a major drive of atherosclerosis and is largely mediated by the NF-kappaB signaling pathway in the vascular and pro-inflammatory cells, notably macrophages. Recently, Sam68, an adaptor protein and Src kinase substrate, has been shown to promote NF-kappaB signaling in mouse embryonic fibroblasts (MEFs). However, whether Sam68 plays a role in the development of atherosclerosis is currently unknown.

Methods and Results: To evaluate the role of Sam68 in atherosclerosis, we crossed Sam68(+/-)mice (Sam68-/- mice are sterile) with ApoE(-/-)mice and obtained ApoE(-/-)Sam68(+/-)and ApoE(-/-)sam68(+/+)mice, which are both grossly normal. Fed on a regular chow, time-course analyses of atherosclerotic lesions were performed histologically throughout the aortas in these mice. ApoE(-/-)Sam68(+/-) mice displayed a significantly delayed development of atheromatous plaques as compared to ApoE(-/-)sam68(+/+)mice at 5, 7, and 10 months of age. The expression levels of pro-inflammatory factors, including TNFalpha, IL-1beta, and IL-6, in the aortas were significantly lower in ApoE(-/-)Sam68(+/-)mice than in ApoE(-/-)Sam68(+/+) mice. Consistently, in the cultured Raw264.7 macrophages, knockdown of Sam68 resulted in a significant reduction in the TNFalpha-induced expression of these pro-inflammatory genes and also in the level of nuclear phospho-p65, indicating an attenuated NF-kappaB activity. Similar results were reproduced in primary macrophages that were isolated from peritoneum or differentiated from bone-marrow mononuclear cells of Sam68(-/-) mice vs. wild-type mice.

Conclusions: Our results for the first time suggest that Sam68 is a critical component of the pro-inflammatory NF-kappaB signaling pathway in macrophages and modulates the course of atherosclerosis at least in ApoE(-/-) mice. Additionally, we have performed Sam68 co-immunoprecipitation and identified by mass spectrometry a panel of new Sam68-interacting proteins. Further experiments are under way to validate those Sam68-interacting proteins that contribute to the NF-kappaB signaling pathway.