Mammalian apolipoprotein B (apo B) exists in two forms, each the product of a single gene. The shorter form, apo B48, arises by posttranscriptional RNA editing whereby cytidine deamination produces a UAA termination codon. A full-length complementary DNA clone encoding an apo B messenger RNA editing protein (REPR) was isolated from rat small intestine. The 229-residue protein contains consensus phosphorylation sites and leucine zipper domains. HepG2 cell extracts acquire editing activity when mixed with REPR from oocyte extracts. REPR is essential for apo B messenger RNA editing, and the isolation and characterization of REPR may lead to the identification of other eukaryotic RNA editing proteins.

Objective To examine infiltration of blood foamy monocytes, containing intracellular lipid droplets, into early atherosclerotic lesions and its contribution to development of nascent atherosclerosis. Approach and Results In apoE−/− mice fed western high-fat diet (WD), >10% of circulating monocytes became foamy monocytes at 3 days on WD and >20% of monocytes at 1 week. Foamy monocytes also formed early in blood of Ldlr−/−Apobec1−/− (LDb) mice on WD. Based on CD11c and CD36, mouse monocytes were categorized as CD11c−CD36−, CD11c−CD36+ and CD11c+CD36+. The majority of foamy monocytes were CD11c+CD36+, whereas most nonfoamy monocytes were CD11c−CD36− or CD11c−CD36+ in apoE−/− mice on WD. In wild-type mice, CD11c+CD36+ and CD11c−CD36+, but few CD11c−CD36−, monocytes took up cholesteryl ester–rich very-low-density lipoproteins (CE-VLDLs) isolated from apoE−/− mice on WD, and CE-VLDL uptake accelerated CD11c−CD36+–to–CD11c+CD36+ monocyte differentiation. Ablation of CD36 decreased monocyte uptake of CE-VLDLs. Intravenous injection of DiI-CE-VLDLs in apoE−/− mice on WD specifically labeled CD11c+CD36+ foamy monocytes, which infiltrated into nascent atherosclerotic lesions and became CD11c+ cells that were selectively localized in atherosclerotic lesions. CD11c deficiency reduced foamy monocyte infiltration into atherosclerotic lesions. Specific and consistent depletion of foamy monocytes (for 3 weeks) by daily intravenous injections of low-dose clodrosome reduced development of nascent atherosclerosis. Conclusions Foamy monocytes, which form early in blood of mice with hypercholesterolemia, infiltrate into early atherosclerotic lesions in a CD11c-dependent manner and play crucial roles in nascent atherosclerosis development.

Objective-Proprotein convertase subtilisin/kexin type 9 (PCSK9) negatively regulates the low-density lipoprotein (LDL) receptor (LDLR) in hepatocytes and therefore plays an important role in controlling circulating levels of LDL-cholesterol. To date, the relationship between PCSK9 and metabolism of apolipoprotein B (apoB), the structural protein of LDL, has been controversial and remains to be clarified. Methods and Results-We assessed the impact of PCSK9 overexpression (≈400-fold above baseline) on apoB synthesis and secretion in 3 mouse models: wild-type C57BL/6 mice and LDLR-null mice (Ldlr −/− and Ldlr). Irrespective of LDLR expression, mice transduced with the PCSK9 gene invariably exhibited increased levels of plasma cholesterol, triacylglycerol, and apoB. Consistent with these findings, the levels of very-low-density lipoprotein and LDL were also increased whereas high-density lipoprotein levels were unchanged. Importantly, we demonstrated that endogenous PCSK9 interacted with apoB in hepatocytes. The PCSK9/apoB interaction resulted in increased production of apoB, possibly through the inhibition of intracellular apoB degradation via the autophagosome/lysosome pathway. Conclusion-We propose a new role for PCSK9 that involves shuttling between apoB and LDLR. The present study thus provides new insights into the action of PCSK9 in regulating apoB metabolism.

SUMMARY Patients with systemic autoimmune diseases show increased incidence of atherosclerosis. However, the contribution of proatherogenic factors to autoimmunity remains unclear. We found that atherogenic mice (herein referred to as LDb mice) exhibited increased serum interleukin-17 (IL-17), which was associated with increased numbers of Th17 cells in secondary lymphoid organs. The environment within LDb mice was substantially favorable for Th17 cell polarization of autoreactive T cells during homeostatic proliferation, and this was substantially inhibited by antibodies directed against oxidized LDL (oxLDL). Moreover, the uptake of oxLDL induced dendritic cell-mediated Th17 polarization by triggering IL-6 production in a process dependent on TLR4, CD36, and MyD88. Furthermore, self-reactive CD4+ T cells expanded in the presence of oxLDL induced more profound experimental autoimmune encephalomyelitis. These findings demonstrate that proatherogenic factors promote the polarization and inflammatory function of autoimmune Th17 cells, which may be critical for the pathogenesis of atherosclerosis and other related autoimmune diseases.

Although complement activation and deposition have been associated with a variety of glomerulopathies, the pathogenic mechanisms by which complement directly mediates renal injury remain to be fully elucidated. Renal parenchymal tissues express a limited repertoire of receptors that directly bind activated complement proteins. We report the renal expression of the receptor for the C3 cleavage product C3a, a member of the anaphylatoxin family. C3aR is highly expressed in normal human and murine kidney, as demonstrated by immunohistochemistry and in situ hybridization. Its distribution is limited to epithelial cells only, as glomerular endothelial and mesangial cells showed no evidence of C3aR expression. The C3aR is also expressed by primary renal proximal tubular epithelial cells in vitro as demonstrated by FACS, Western blot, and RT-PCR. In vitro C3aR is functional in terms of its capacity to bind 125I-labeled C3a and generate inositol triphosphate. Finally, using microarray analysis, four novel genes were identified and confirmed as transcriptionally regulated by C3aR activation in proximal tubular cells. These studies define a new pathway by which complement activation may directly modulate the renal response to immunologic injury.

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Atherosclerosis, a deadly disease insufficiently addressed by cholesterol-lowering drugs, needs new therapeutic strategies. Fortilin, a 172-amino acid multifunctional polypeptide, binds p53 and blocks its transcriptional activation of Bax, thereby exerting potent antiapoptotic activity. Although fortilin-overexpressing mice reportedly exhibit hypertension and accelerated atherosclerosis, it remains unknown if fortilin, not hypertension, facilitates atherosclerosis. Our objective was to test the hypothesis that fortilin in and of itself facilitates atherosclerosis by protecting macrophages against apoptosis. We generated fortilin-deficient ( fortilin+/−) mice and wild-type counterparts ( fortilin+/+) on a LDL receptor ( Ldlr)−/− apolipoprotein B mRNA editing enzyme, catalytic polypeptide 1 ( Apobec1)−/− hypercholesterolemic genetic background, incubated them for 10 mo on a normal chow diet, and assessed the degree and extent of atherosclerosis. Despite similar blood pressure and lipid profiles, fortilin+/− mice exhibited significantly less atherosclerosis in their aortae than their fortilin +/+ littermate controls. Quantitative immunostaining and flow cytometry analyses showed that the atherosclerotic lesions of fortilin+/− mice contained fewer macrophages than those of fortilin+/+ mice. In addition, there were more apoptotic cells in the intima of fortilin+/− mice than in the intima of fortilin+/+ mice. Furthermore, peritoneal macrophages from fortilin+/− mice expressed more Bax and underwent increased apoptosis, both at the baseline level and in response to oxidized LDL. Finally, hypercholesterolemic sera from Ldlr−/− Apobec1−/− mice induced fortilin in peritoneal macrophages more robustly than sera from control mice. In conclusion, fortilin, induced in the proatherosclerotic microenvironment in macrophages, protects macrophages against Bax-induced apoptosis, allows them to propagate, and accelerates atherosclerosis. Anti-fortilin therapy thus may represent a promising next generation antiatherosclerotic therapeutic strategy.

APOBEC-1 is a catalytic subunit of an apolipoprotein B (apoB) mRNA editing enzyme complex. In humans it is expressed only in the intestine, whereas in mice it is expressed in both the liver and intestine. APOBEC-1 exists as a spontaneous homodimer (Lau, P. P., Zhu, H.-J., Baldini, A., Charnsangavej, C., and Chan, L. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 8522-8526). We tested the editing activity and dimerization potential of three different mouse APOBEC-1 mutants using in vitro editing activity assay and immunoprecipitation in the presence of epitope-tagged APOBEC-1. One catalytically inactive mutant, mu1 (H61K/C93S/C96S), that retains its capacity to dimerize with wild-type APOBEC-1 was found to inhibit the editing activity of the latter and was thus a dominant negative mutant. Two other inactive mutants that dimerized poorly with APOBEC-1 failed to inhibit its activity. Intravenous injection of a mu1 adenovirus, Admu1, in C57BL/6J mice in vivo resulted in liver-specific expression of mu1 mRNA. On days 4 and 9 after virus injection, endogenous hepatic apoB mRNA editing was 23.3 ؎ 5.0 and 36.8 ؎ 5.7%, respectively, compared with 65.3 ؎ 11 and 71.3 ؎ 5.2%, respectively, for luciferase adenovirus-treated animals. Plasma apoB-100 accounted for 95 and 93% of total plasma apoB in Admu1 animals on days 4 and 9, respectively, compared with 78 and 72% in luciferase adenovirus animals. Plasma cholesterol on day 9 was 98 ؎ 17 mg/dl in the mu1-treated animals, substantially higher than phosphate-buffered saline-treated (57 ؎ 9 mg/dl) or luciferase-treated (71 ؎ 12 mg/dl) controls. Fast protein liquid chromatography analysis of mouse plasma showed that the intermediate density/low density lipoprotein fractions in the animals treated with the dominant negative mutant adenovirus were much higher than those in controls. We conclude that active APOBEC-1 functions as a dimer and its activity is inhibited by a dominant negative mutant. Furthermore, apoB mRNA editing determines the availability of apoB-100, which in turn limits the amount of intermediate density/low density lipoprotein that can be formed in mice. Liver-specific inhibition of apoB mRNA editing is an important component of any strategy to enhance the value of mice as a model for human lipoprotein metabolism.The mouse is a useful animal model for lipoprotein metabolism and atherosclerosis (1, 2). Its value as a model for human disease, however, is limited by the fact that there is a substantial difference in lipoprotein metabolism between the two species. One major difference is the presence of high levels of apolipoprotein B (apoB) 1 mRNA editing in the liver in mice but not in humans.ApoB mRNA editing is a process by which apoB-100 mRNA is converted to apoB-48 mRNA (3, 4) (reviewed in Ref. 5). It involves the conversion of the first base of the codon CAA, encoding glutamine 2153 in apoB-100, to UAA, a stop codon, in apoB-48 mRNA. In humans, editing occurs exclusively in the small intestine but not in the liver. Therefore, the human liver produces apoB-100 and ...