Activation of the Unfolded Protein Response Occurs at All Stages of Atherosclerotic Lesion Development in Apolipoprotein E–Deficient MiceBACKGROUND: Apoptotic cell death contributes to atherosclerotic lesion instability, rupture, and thrombogenicity. Recent findings suggest that free cholesterol (FC) accumulation in macrophages induces endoplasmic reticulum (ER) stress/unfolded protein response (UPR) and apoptotic cell death; however, it is not known at what stage of lesion development the UPR is induced in macrophages or whether a correlation exists between UPR activation, FC accumulation, and apoptotic cell death. METHODS AND RESULTS: Aortic root sections from apolipoprotein E-deficient (apoE-/-) mice at 9 weeks of age (early-lesion group) or 23 weeks of age (advanced-lesion group) fed a standard chow diet were examined for markers of UPR activation (GRP78, phospho-PERK, CHOP, and TDAG51), apoptotic cell death (TUNEL and cleaved caspase-3), and lipid accumulation (filipin and oil red O). UPR markers were dramatically increased in very early intimal macrophages and in macrophage foam cells from fatty streaks and advanced atherosclerotic lesions. Although accumulation of FC was observed in early-lesion-resident macrophage foam cells, no evidence of apoptotic cell death was observed; however, UPR activation, FC accumulation, and apoptotic cell death were observed in a small percentage of advanced-lesion-resident macrophage foam cells. CONCLUSIONS: UPR activation occurs at all stages of atherosclerotic lesion development. The additional finding that macrophage apoptosis did not correlate with UPR activation and FC accumulation in early-lesion-resident macrophages suggests that activation of other cellular mediators and/or pathways are required for apoptotic cell death.
Induction of the unfolded protein response after monocyte to macrophage differentiation augments cell survival in early atherosclerotic lesionsABSTRACT Endoplasmic reticulum (ER) stress causes macrophage cell death within advanced atherosclerotic lesions, thereby contributing to necrotic core formation and increasing the risk of atherothrombotic disease. However’ unlike in advanced lesions’ the appearance of dead/apoptotic macrophages in early lesions is less prominent. Given that activation of the unfolded protein response (UPR) is detected in early lesion‐resident macrophages and can enhance cell survival against ER stress’ we investigated whether UPR activation occurs after monocyte to macrophage differentiation and confers a cytoprotective advantage to the macrophage. Human peripheral blood monocytes were treated with monocyte colony‐stimulating factor to induce macrophage differentiation’ as assessed by changes in ultrastructure and scavenger receptor expression. UPR markers, including GRP78, GRP94, and spliced XBP‐1, were induced after macrophage differentiation and occurred after a significant increase in de novo protein synthesis. UPR activation after differentiation reduced macrophage cell death by ER stress‐inducing agents. Further, GRP78 overexpression in macrophages was sufficient to reduce ER stress‐induced cell death. Consistent with these in vitro findings, UPR activation was observed in viable lesion‐resident macrophages from human carotid arteries and from the aortas of apoE _/_ mice. However, no evidence of apoptosis was observed in early lesion‐resident macrophages from the aortas of apoE _/_ mice. Thus, our findings that UPR activation occurs during macrophage differentiation and is cyto‐protective against ER stress‐inducing agents suggest an important cellular mechanism for macrophage survival within early atherosclerotic lesions.—Dickhout, J. G., Lhotak, S., Hilditch, B. A., Basseri, S., Colgan, S. M., Lynn, E. G., Carlisle, R. E., Zhou, J., Sood, S. K., Ingram, A. J., Austin, R. C. Induction of the unfolded protein response after monocyte to macrophage differentiation augments cell survival in early atherosclerotic lesions. FASEB J. 25, 576–589 (2011). www.fasebj.org
Macrophage Differentiation Involves Activation of the Unfolded Protein ResponseTo determine if the unfolded protein response (UPR) is activated during macrophage differentiation and is cytoprotective to macrophages in atherosclerosis, human peripheral blood monocytes were treated with macrophage colony stimulating factor to induce differentiation. Differentiation was assessed as well as UPR activation. UPR markers upregulated during differentiation included the protein folding chaperones GRP78, GRP94 and calnexin and XBP‐1 mRNA splicing. However, the expression of these UPR markers was temporally distinct from ER stress‐induced UPR, did not saturate as further induction occurred with ER stress and did not involve oxidative stress. A dramatic increase in protein synthesis (31‐fold), measured by 35 S‐ methionine incorporation, preceded UPR activation. Cytoprotection against ER stress followed UPR activation or plasmid‐mediated GRP78 overexpression. In early atherosclerotic lesions of apoE −/− mice, GRP78 was markedly increased in resident macrophages, but not monocytes. Our findings demonstrate that UPR activation occurs as a physiological response to increased protein synthesis during macrophage differentiation and is cytoprotective. This may represent an important mechanism for macrophage survival in atherosclerotic lesions, contributing to lesion development and progression. Supported by the Heart and Stroke Foundation of Ontario (T‐5385) and the CIHR (MOP‐74477).