Background-Macrophage apoptosis is a critical process in the formation of necrotic cores in vulnerable atherosclerotic plaques. In vitro and in vivo data suggest that macrophage apoptosis in advanced atheromata may be triggered by a combination of endoplasmic reticulum stress and engagement of the type A scavenger receptor, which together induce death through a rise in cytosolic calcium and activation of toll-like receptor-4. Methods and Results-Using both primary peritoneal macrophages and studies in advanced atheromata in vivo, we introduce signal transducer and activator of transcription-1 (STAT1) as a critical and necessary component of endoplasmic reticulum stress/type A scavenger receptor-induced macrophage apoptosis. We show that STAT1 is serine phosphorylated in macrophages subjected to type A scavenger receptor ligands and endoplasmic reticulum stress in a manner requiring cytosolic calcium, calcium/calmodulin-dependent protein kinase II, and toll-like receptor-4. Remarkably, apoptosis was inhibited by Ϸ80% to 90% (PϽ0.05) by STAT1 deficiency or calcium/calmodulin-dependent protein kinase II inhibition. In vivo, nuclear Ser-P-STAT1 was found in macrophage-rich regions of advanced murine and human atheromata. Most important, macrophage apoptosis was decreased by 61% (Pϭ0.034) and plaque necrosis by 34% (Pϭ0.02) in the plaques of fat-fed low density lipoprotein receptor null Ldlr Ϫ/Ϫ mice transplanted with Stat1 Ϫ/Ϫ bone marrow. Conclusions-STAT1 is critical for endoplasmic reticulum stress/type A scavenger receptor-induced apoptosis in primary tissue macrophages and in macrophage apoptosis in advanced atheromata. These findings suggest a potentially important role for STAT1-mediated macrophage apoptosis in atherosclerotic plaque progression. Key Words: apoptosis Ⅲ atherosclerosis Ⅲ cholesterol Ⅲ macrophage Ⅲ plaque I n advanced atherosclerosis, death of macrophages in the setting of defective phagocytic clearance of apoptotic cells contributes to the development of plaque necrosis. 1,2 Plaque necrosis, in turn, is thought to promote plaque disruption and arterial thrombosis, which are the proximate causes of acute cardiovascular events. 1-3 Our laboratory established an important principle of advanced lesional macrophage death, namely involvement of the endoplasmic reticulum (ER) stress pathway known as the unfolded protein response (UPR). 4,5 Some laboratories have discovered important evidence that the UPR is activated in intimal cells, including macrophages, in advanced murine and human plaques. 6 -9 In particular, Myoishi et al 9 recently showed a dramatic rise in UPR markers, including the transcription factor CHOP (GADD153), and intimal cell apoptosis in autopsy specimens from humans with vulnerable and ruptured plaques but not stable lesions and in atherectomy specimens from humans with unstable angina but not stable angina. Although the UPR is primarily an ER repair pathway, a branch of the UPR involving the effector CHOP can trigger apoptosis when the cell senses that repair is no longer poss...

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Plaque necrosis in advanced atheromata, which triggers acute atherothrombotic vascular events, is caused by the apoptosis of lesional macrophages coupled with defective phagocytic clearance of the dead cells. The central enabling event in macrophage apoptosis relevant to advanced atherosclerosis is the unfolded protein response (UPR), an endoplasmic reticulum (ER) stress pathway. The UPR effector CHOP (GADD153) amplifies release of ER Ca2+ stores, which activates a central integrator of apoptosis signaling, calcium/calmodulin-dependent protein kinase II (CaMKII). CaMKII, in turn, leads to activation of pro-apoptotic STAT1, induction of the death receptor Fas, and stimulation of the mitochondria-cytochrome c pathway of apoptosis. While these pathways are necessary for apoptosis, apoptosis occurs only when the cells are also exposed to one or more additional “hits.” These hits amplify pro-apoptotic pathways and/or suppress compensatory cell-survival pathways. A second hit relevant to atherosclerosis is activation of pattern recognition receptors (PRRs), such as scavenger and toll-like receptors. In vivo relevance is suggested by the fact that advanced human lesions express markers of UPR activation that correlate closely with the degree of plaque vulnerability and macrophage apoptosis. Moreover, studies with genetically altered mice have shown that ER stress and PRR activation are causative for advanced lesional macrophage apoptosis and plaque necrosis. In summary, a key cellular event in the conversion of benign to vulnerable atherosclerotic plaques is ER stress-induced macrophage apoptosis. Further understanding of the mechanisms and consequences of this event may lead to novel therapies directed at preventing the clinical progression of atheromata.

CPX-351, a liposomal formulation co-encapsulating cytarabine (Cyt) and daunorubicin (Daun), has been developed, which delivers synergistic Cyt:Daun molar ratios to bone marrow. CPX-351 has demonstrated markedly superior anti-leukemic activity over free Cyt:Daun drug cocktails in preclinical models. Given the prolonged plasma lifetime of CPX-351, we examined the relationship between therapeutic efficacy and the frequency of treatment in the consolidation setting using a bone marrow-engrafting human leukemia xenograft model. Adding a day 1,3,5 consolidation treatment course for CPX-351 therapy improved the increase in lifespan (ILS) from 116% and no cures for a single induction course, to 268% plus a 33% cure rate for an induction plus consolidation course. In contrast, free Cyt:Daun cocktail treatment provided much lower ILS values with no cures. Administering CPX-351 as consolidation therapy starting on day 42 using a day 1,3, day 1,5, or day 1,7 schedule yielded ILS values of 154%, 185%, and 108%, respectively. The increased efficacy observed for the day 1,3 and day 1,5 consolidation schedules was associated with elevated bone marrow drug accumulation for the second doses. The enhanced efficacy obtained for intermediate dosing frequency in the consolidation setting suggests that the anti-leukemic activity of synergistic drug ratios is dependent on both duration of exposure and maintenance above a therapeutic threshold.