Rationale Human atherosclerotic plaques contain large numbers of cells deprived of O2. In murine atherosclerosis, because the plaques are small, it is controversial whether hypoxia can occur. Objective To examine if murine plaques contain hypoxic cells, and whether hypoxia regulates changes in cellular lipid metabolism and gene expression in macrophages. Methods and Results Aortic plaques from apolipoprotein-E-deficient mice were immunopositive for hypoxia-inducible transcription factor (HIF-1α) and some of its downstream targets. Murine J774 macrophages rendered hypoxic demonstrated significant increases in cellular sterol and triglycerides. The increase in sterol content in hypoxic macrophages correlated with elevated 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase activity and mRNA levels. In addition, when macrophages were incubated with cholesterol complexes, hypoxic cells accumulated 120% more cholesterol, predominately in the free form. Cholesterol-efflux assays showed that hypoxia significantly decreased efflux mediated by ATP binding cassette sub-family A member 1 (ABCA1), whose sub cellular localization was altered in both J774 and primary macrophages. Furthermore, in vivo expression patterns of selected genes from cells in hypoxic regions of murine plaques were similar to those from J774 and primary macrophages incubated in hypoxia. The hypoxia-induced accumulation of sterol and decreased cholesterol efflux was substantially reversed in vitro by reducing the expression of the hypoxia-inducible transcription factor, HIF-1α. Conclusion Hypoxic regions are present in murine plaques. Hypoxic macrophages have increased sterol content due to the induction of sterol synthesis and the suppression of cholesterol efflux, effects that are in part mediated by HIF-1α.

Efficacy and safety of iron chelation therapy with deferasirox in iron-overloaded non-transfusion-dependent thalassaemia (NTDT) patients were established in the THALASSA study. THETIS, an open-label, single-arm, multicentre, Phase IV study, added to this evidence by investigating earlier dose escalation by baseline liver iron concentration (LIC) (week 4: escalation according to baseline LIC; week 24: adjustment according to LIC response, maximum 30mg/kg/day). The primary efficacy endpoint was absolute change in LIC from baseline to week 52. 134 iron-overloaded non-transfusion-dependent anaemia patients were enrolled and received deferasirox starting at 10mg/kg/day. Mean actual dose±SD over 1year was 14.70±5.48mg/kg/day. At week 52, mean LIC±SD decreased significantly from 15.13±10.72mg Fe/g dw at baseline to 8.46±6.25mg Fe/g dw (absolute change from baseline, -6.68±7.02mg Fe/g dw [95% CI: -7.91, -5.45]; P<0.0001). Most common drug-related adverse events were gastrointestinal: abdominal discomfort, diarrhoea and nausea (n=6 each). There was one death (pneumonia, not considered drug related). With significant and clinically relevant reductions in iron burden alongside a safety profile similar to that in THALASSA, these data support earlier escalation with higher deferasirox doses in iron-overloaded non-transfusion-dependent anaemia patients.

We have previously reported enhancing the imaging of atherosclerotic plaques in mice using reconstituted high density lipoproteins (HDL) as nanocarriers for the MRI contrast agent gadolinium (Gd). This study focuses on the underlying mechanisms of Gd delivery to atherosclerotic plaques. HDL, LDL, and VLDL particles containing Gd chelated to phosphatidyl ethanolamine (DTPA-DMPE) and a lipidic fluorophore were used to demonstrate the transfer of Gd-phospholipids among plasma lipoproteins in vitro and in vivo. To determine the basis of this transfer, the roles of phospholipid transfer protein (PLTP) and lipoprotein lipase (LpL) in mediating the migration of Gd-DTPA-DMPE among lipoproteins were investigated. The results indicated that neither was an important factor, suggesting that spontaneous transfer of Gd-DTPA-DMPE was the most probable mechanism. Finally, two independent mouse models were used to quantify the relative contributions of HDL and LDL reconstituted with Gd-DTPA-DMPE to plaque imaging enhancement by MR. Both sets of results suggested that Gd-DTPA-DMPE originally associated with LDL was about twice as effective as that injected in the form of Gd-HDL, and that some of Gd-HDL’s effectiveness in vivo is indirect through transfer of the imaging agent to LDL. In conclusion, the fate of Gd-DTPA-DMPE associated with a particular type of lipoprotein is complex, and includes its transfer to other lipoprotein species that are then cleared from the plasma into tissues.

Although esterification of free cholesterol to cholesteryl ester in the liver is known to be catalyzed by the enzyme acyl-coenzyme A:cholesterol acyltransferase, ACAT, the neutral cholesteryl ester hydrolase (nCEH) that catalyzes the reverse reaction has remained elusive. Because cholesterol undergoes continuous cycling between free and esterified forms, the steady-state concentrations in the liver of the two species and their metabolic availability for pathways, such as lipoprotein assembly and bile acid synthesis, depend upon nCEH activity. On the basis of the general characteristics of the family of rat carboxylesterases, we hypothesized that one member, ES-4, was a promising candidate as a hepatic nCEH. Using under-and overexpression approaches, we provide multiple lines of evidence that establish ES-4 as a bona fide endogenous nCEH that can account for the majority of cholesteryl ester hydrolysis in transformed rat hepatic cells and primary rat hepatocytes.Cytosolic cholesterol pools are in a dynamic state of turnover between the free (FC) 2 and esterified forms (CE) (1-3). The identification of the enzyme(s) in the liver that catalyzes the hydrolysis of CE at neutral pH has been elusive. In contrast, the CE that enters cells as part of apoB-containing lipoproteins through receptor-or heparan sulfate proteoglycan-mediated uptake is known to be hydrolyzed by lysosomal acid lipase, with the ensuing FC available to enter various cellular pools. A fraction of the FC trafficked to the endoplasmic reticulum (ER) is esterified by isoforms of acyl-coenzyme A:cholesterol acyltransferase (ACAT). When potentially cytotoxic levels of FC are reached, there is a significant increase in the formation of CE, which is then stored in cytosolic lipid droplets (4, 5).In the liver, CE is a component of very low density lipoproteins (VLDL). The source of CE can be either direct (i.e. synthesized completely in the ER membrane) or indirect through hydrolysis of cytosolic CE and re-esterification of FC by ACAT (6, 7). FC can also become part of VLDL, and, as in all cells, FC resulting from CE hydrolysis can be effluxed to HDL or used in the synthesis of other molecules. In liver, a major fate of FC is its conversion to bile acids and oxysterols. The interconversion between the non-lysosomal cellular pools of FC and CE in the liver is under metabolic regulation, with the key enzymatic components attributed to ACAT and neutral cholesteryl ester hydrolases (nCEHs) (8 -12).The explicit identification of nCEHs in the liver has been elusive. Ghosh and colleagues (13) have recently reported a human macrophage nCEH active in foam cells in atherosclerotic plaques that was related to a previously studied enzyme proposed as a rat hepatic nCEH (14). The latter was cloned from rat liver, and, when overexpressed in COS cells, the lysates exhibited nCEH activity against exogenously provided CE (14). However, whether this candidate operates as a nCEH under physiological conditions in hepatic cells has never been established. Another candidate that...