Genetic and dietary regulations of autophagy activity and cholesterol homeostasis in macrophage foam cells
Zahid, MD Khurshidul
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Background: Atherosclerosis which is associated with deregulated cholesterol metabolism and formation of macrophage foam cells which is a major cause of mortality and morbidity in western societies. These foam cells can stimulate the production of inflammatory cytokines, induce endoplasmic reticulum stress (ERS) and apoptosis and ultimately accelerate atherosclerosis development. Autophagy is a catabolic pathway and has been shown to regulate cholesterol homeostasis in atherosclerosis. CCAAT/enhancer binding protein beta (C/EBPβ) is a transcription factor and its inhibition has recently been shown to prevent atherosclerosis development and foam cell formation. However, whether C/EBPβ beta regulates ERS, apoptosis, and autophagy in atherosclerosis remains unknown. Blueberry is a polyphenol-rich fruit with known health benefits. In contrast, less is known about the physiological functions of bioactive compounds present in blueberry leaf (BBL). We investigated the role of C/EBP beta inhibition in regulating ERS, apoptosis, and autophagy in macrophage foam cells. In addition, we also investigated the effects of BBL extract on foam cell formation, autophagy, and cholesterol efflux in lipid loaded macrophages. Methods and materials: For the first set of experiments, RAW 264.7 macrophage cells were transfected with control and CEBP β -siRNA (50 nM) for 24h to knockdown C/EBPβ and treated with nLDL and OXLDL (25μg/ml) for an additional 24 hr. Then key proteins and genes involved in cholesterol efflux (ABCA1, ABCG1), autophagy (LC3, ATG5), inflammation (p-NFkB), ER stress (ATF4, ATF6), and apoptosis (CHOP, Caspase 1, Caspase 3 and Caspase 12) were analyzed by western blot and qPCR. In second set of experiment, RAW 264.7 and bone marrow-derived macrophages (BMDM) were treated without (control) and with LPS (100 ng/ml) and oxLDL (25 µg/ml) for 6-24 h followed by treatment with BBL (10 and 25µg/ml) for an additional 6-24 h. Expression of proteins and genes were analyzed by western blot and QRT-PCR respectively. Oil Red O staining was employed to detect lipid accumulation. Autophagy vacuoles were also detected in macrophages by autophagy detection kit. Results: We found that short interfering RNA mediated knockdown of C/EBPβ attenuated atherogenic lipid-mediated induction of proteins implicated in inflammation (NFkB-p65), ERS (activating transcription factor 4 and 6 (ATF4 and ATF6)) and apoptosis genes and protein (CHOP, caspase 3, and 12) expression. Interestingly, C/EBPβ knockdown up-regulated autophagy (LC3II, ATG5) and decreased mTOR protein phosphorylation, in lipid-loaded macrophages. C/EBPβ knockdown also increased cholesterol efflux (ABCG1), autophagy gene (ATG5, LC3B) while decreased mTORC1, apoptotic (CHOP, caspase1, 3, 12), and ERS (ATF4 and ATF6) gene expression. Blueberry leaf (BBL) extract attenuated LPS and oxLDL mediated induction of foam cell formation in RAW macrophage cells. Interestingly, BBL extract increased the expressions of autophagy proteins (LC3II and ATG5 and autophagic vacuoles. This was associated with increased ABCA1 protein expression (which is involved in cholesterol efflux). In addition, BBL extract increased phosphorylated form of AMPK and decreased mTORC1, ATF4, and CHOP gene expression. Conclusion: The present results suggest that both C/EBPβ inhibition and BBL extract regulate inflammation, ERS and apoptosis in macrophage foam cells in part by increasing autophagy. The results also suggest that C/EBPβ and blueberry leaf (BBL) extract promote cholesterol efflux and autophagy activity via AMPK/mTOR signaling in macrophage foam cells. Thus, inhibition of C/EBPβ and/ or use of BBL extract may be important therapeutic options to prevent foam cell formation and atherosclerosis development.