HDL cholesterol (HDL-C) plasma levels are inversely related to cardiovascular disease risk. Previous studies have shown in animals and humans that HDL promotes regression of atherosclerosis. We ...hypothesized that this was related to an ability to promote the loss of monocyte-derived cells (CD68âº, primarily macrophages and macrophage foam cells) from plaques. To test this hypothesis, we used an established model of atherosclerosis regression in which plaque-bearing aortic arches from apolipoprotein E-deficient (apoEâ»/â») mice (low HDL-C, high non-HDL-C) were transplanted into recipient mice with differing levels of HDL-C and non-HDL-C: C57BL6 mice (normal HDL-C, low non-HDL-C), apoAIâ»/â» mice (low HDL-C, low non-HDL-C), or apoEâ»/â» mice transgenic for human apoAI (hAI/apoEâ»/â»; normal HDL-C, high non-HDL-C). Remarkably, despite persistent elevated non-HDL-C in hAI/apoEâ»/â» recipients, plaque CD68⺠cell content decreased by >50% by 1 wk after transplantation, whereas there was little change in apoAIâ»/â» recipient mice despite hypolipidemia. The decreased content of plaque CD68⺠cells after HDL-C normalization was associated with their emigration and induction of their chemokine receptor CCR7. Furthermore, in CD68⺠cells laser-captured from the plaques, normalization of HDL-C led to decreased expression of inflammatory factors and enrichment of markers of the M2 (tissue repair) macrophage state. Again, none of these beneficial changes were observed in the apoAIâ»/â» recipients, suggesting a major requirement for reverse cholesterol transport for the beneficial effects of HDL. Overall, these results establish HDL as a regulator in vivo of the migratory and inflammatory properties of monocyte-derived cells in mouse atherosclerotic plaques, and highlight the phenotypic plasticity of these cells.
In contrast to studies on class I histone deacetylase (HDAC) inhibitors, the elucidation of the molecular mechanisms and therapeutic potential of class IIa HDACs (HDAC4, HDAC5, HDAC7 and HDAC9) is ...impaired by the lack of potent and selective chemical probes. Here we report the discovery of inhibitors that fill this void with an unprecedented metal-binding group, trifluoromethyloxadiazole (TFMO), which circumvents the selectivity and pharmacologic liabilities of hydroxamates. We confirm direct metal binding of the TFMO through crystallographic approaches and use chemoproteomics to demonstrate the superior selectivity of the TFMO series relative to a hydroxamate-substituted analog. We further apply these tool compounds to reveal gene regulation dependent on the catalytic active site of class IIa HDACs. The discovery of these inhibitors challenges the design process for targeting metalloenzymes through a chelating metal-binding group and suggests therapeutic potential for class IIa HDAC enzyme blockers distinct in mechanism and application compared to current HDAC inhibitors.
Mouse aortic smooth muscle cells (SMCs) were loaded for 72 h with cholesterol by using cholesterol:methyl-β-cyclodextrin complexes, leading to ≈2-fold and ≈10-fold increases in the contents of total ...cholesterol and cholesteryl ester, respectively. Foam-cell formation was demonstrated by accumulation of intracellular, Oil Red O-stained lipid droplets. Immunostaining showed decreased protein levels of smooth muscle α-actin and α-tropomyosin and increased levels of macrophage markers CD68 and Mac-2 antigen. Quantitative real-time RT-PCR revealed that after cholesterol loading, the expression of SMC-related genes α-actin, α-tropomyosin, myosin heavy chain, and calponin H1 decreased (to 11.5 ± 0.5%, 29.3 ± 1.4%, 23.8 ± 1.4%, and 3.8 ± 0.5% of unloaded cells, respectively; P < 0.05 for all), whereas expression of macrophage-related genes CD68, Mac-2, and ABCA1 mRNA increased (to 709 ± 84%, 330 ± 11%, and 207 ± 13% of unloaded cells, respectively; P < 0.05 for all), thereby demonstrating that the protein changes were regulated at the mRNA level. Furthermore, these changes were accompanied by a gain in macrophage-like function as assessed by phagocytotic activity. Expression of vascular cell adhesion molecule 1 and monocyte chemoattractant protein 1, known responders to inflammation, were not changed. In conclusion, cholesterol loading of SMC causes phenotypic changes regulated at the mRNA level that result in a transdifferentiation to a macrophage-like state. This finding suggests that not all foam cells in lesions may have a macrophage origin, despite what is indicated by immunostaining for macrophage-related markers. Furthermore, inflammatory changes in foam cells observed in vivo may not be simple consequences of cholesterol accumulation.
We previously showed that the progression of atherosclerosis in the Reversa mouse (Ldlr(-/-Apob100/100Mttpfl/fl) Mx1Cre(+/+)) was arrested when the hyperlipidemia was normalized by inactivating the ...gene for microsomal triglyceride transfer protein. Here, we tested whether atherosclerosis would regress if the lipid levels were reduced after advanced plaques formed.
Reversa mice were fed an atherogenic diet for 16 weeks. Plasma lipid levels were then reduced. Within 2 weeks, this reduction led to decreased monocyte-derived (CD68(+)) cells in atherosclerotic plaques and was associated with emigration of these cells out of plaques. In addition, the fall in lipid levels was accompanied by lower plaque lipid content and by reduced expression in plaque CD68(+) cells of inflammatory genes and higher expression of genes for markers of antiinflammatory M2 macrophages. Plaque composition was affected more than plaque size, with the decreased content of lipid and CD68(+) cells balanced by a higher content of collagen. When the reduced lipid level was combined with the administration of pioglitazone to simulate the clinical aggressive lipid management and proliferator-activated receptor-γ agonist treatment, the rate of depletion of plaque CD68(+) cells was unaffected, but there was a further increase in their expression of antiinflammatory macrophage markers.
The Reversa mouse is a new model of atherosclerosis regression. After lipid lowering, favorable changes in plaque composition were independent of changes in size. In addition, plaque CD68(+) cells became less inflammatory, an effect enhanced by treatment with pioglitazone.
Adipose Mitochondrial Biogenesis Is Suppressed in db/db and High-Fat Diet–Fed Mice and Improved by Rosiglitazone
James X. Rong 1 ,
Yang Qiu 2 3 ,
Michael K. Hansen 3 ,
Lei Zhu 3 4 ,
Vivian Zhang 3 ,
...Mi Xie 1 ,
Yuji Okamoto 5 ,
Michael D. Mattie 6 ,
Hiroyuki Higashiyama 5 ,
Satoshi Asano 5 ,
Jay C. Strum 6 and
Terence E. Ryan 3
1 High Throughput Biology, Discovery Research, GlaxoSmithKline, Research Triangle Park, North Carolina
2 Cheminformatics, Discovery Research, GlaxoSmithKline, Research Triangle Park, North Carolina
3 Integrative Biology, High Throughput Biology, Discovery Research, GlaxoSmithKline, King of Prussia, Pennsylvania
4 Biomedical Data Sciences, GlaxoSmithKline, Research Triangle Park, North Carolina
5 Tsukuba Research Laboratories, High Throughput Biology, Discovery Research, GlaxoSmithKline, Ibaraki, Japan
6 Department of Quantitative Expression, Genetics Research, GlaxoSmithKline, Research Triangle Park, North Carolina
Address correspondence and reprint requests to James X. Rong, High Throughput Biology, Discovery Research, GlaxoSmithKline,
5 Moore Dr., Research Triangle Park, NC. E-mail: james.x.rong{at}gsk.com
Abstract
The objective of this study was to further establish and confirm the relationship of adipose mitochondrial biogenesis in diabetes/obesity
and the effects of rosiglitazone (RSG), a peroxisome proliferator–activated receptor (PPAR) γ agonist, by systematically analyzing
mitochondrial gene expression and function in two mouse models of obesity and type 2 diabetes. Using microarray technology,
adipose mitochondrial gene transcription was studied in db/db , high-fat diet–fed C57BL/6 (HFD) and respective control mice with or without RSG treatment. The findings were extended using
mitochondrial staining, DNA quantification, and measurements of citrate synthase activity. In db/db and HFD mice, gene transcripts associated with mitochondrial ATP production, energy uncoupling, mitochondrial ribosomal proteins,
outer and inner membrane translocases, and mitochondrial heat-shock proteins were decreased in abundance, compared with db/+
and standard-fat diet–fed control mice, respectively. RSG dose-dependently increased these transcripts in both db/db and HFD mice and induced transcription of mitochondrial structural proteins and cellular antioxidant enzymes responsible
for removal of reactive oxygen species generated by increased mitochondrial activity. Transcription factors, including PPAR
coactivator (PGC)-1β, PGC-1α, estrogen-related receptor α, and PPARα, were suppressed in both models and induced by RSG. The
effects of RSG on adipose mitochondrial genes were confirmed by quantitative RT-PCR and further supported by mitochondrial
staining, mitochondrial DNA quantification, and citrate synthase activity. Adipose mitochondrial biogenesis was overwhelmingly
suppressed in both mouse models of diabetes/obesity and globally induced by RSG. These findings suggest an important role
of adipose mitochondria in diabetes/obesity and the potential for new treatment approaches targeting adipose mitochondria.
BAT, brown adipose tissue
CIDEA, cell death–inducing DFFA-like effector a
CPT, carnitinepalmitoyl transferase
FAO, fatty acid oxidation
HSP, heat-shock protein
OXPHOS, oxidative phosphorylation
PGC, PPAR coactivator
PPAR, peroxisome proliferator–activated receptor
RIP, receptor interacting protein
RSG, rosiglitazone
TCA, tricarboxylic acid
UCP, uncoupling protein
WAT, white adipose tissue
Footnotes
Published ahead of print at http://diabetes.diabetesjournals.org on 24 April 2007. DOI: 10.2337/db06-1135.
Additional information for this article can be found in an online appendix at http://dx.doi.org/10.2337/db06-1135 .
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore
be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Accepted April 11, 2007.
Received August 14, 2006.
DIABETES
Excess cellular cholesterol induces apoptosis in macrophages, an event likely to promote progression of atherosclerosis. The cellular mechanism of cholesterol-induced apoptosis is unknown but had ...previously been thought to involve the plasma membrane. Here we report that the unfolded protein response (UPR) in the endoplasmic reticulum is activated in cholesterol-loaded macrophages, resulting in expression of the cell death effector CHOP. Cholesterol loading depletes endoplasmic reticulum calcium stores, an event known to induce the UPR. Furthermore, endoplasmic reticulum calcium depletion, the UPR, caspase-3 activation and apoptosis are markedly inhibited by selective inhibition of cholesterol trafficking to the endoplasmic reticulum, and Chop-/- macrophages are protected from cholesterol-induced apoptosis. We propose that cholesterol trafficking to endoplasmic reticulum membranes, resulting in activation of the CHOP arm of the UPR, is the key signalling step in cholesterol-induced apoptosis in macrophages.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
The sensitivity of current surveillance methods for detecting early-stage hepatocellular carcinoma (HCC) is suboptimal. Extracellular vesicles (EVs) are promising circulating biomarkers for early ...cancer detection. In this study, we aim to develop an HCC EV-based surface protein assay for early detection of HCC.
Tissue microarray was used to evaluate four potential HCC-associated protein markers. An HCC EV surface protein assay, composed of covalent chemistry-mediated HCC EV purification and real-time immuno-polymerase chain reaction readouts, was developed and optimized for quantifying subpopulations of EVs. An HCC EV ECG score, calculated from the readouts of three HCC EV subpopulations ( E pCAM + CD63 + , C D147 + CD63 + , and G PC3 + CD63 + HCC EVs), was established for detecting early-stage HCC. A phase 2 biomarker study was conducted to evaluate the performance of ECG score in a training cohort ( n = 106) and an independent validation cohort ( n = 72).Overall, 99.7% of tissue microarray stained positive for at least one of the four HCC-associated protein markers (EpCAM, CD147, GPC3, and ASGPR1) that were subsequently validated in HCC EVs. In the training cohort, HCC EV ECG score demonstrated an area under the receiver operating curve (AUROC) of 0.95 (95% confidence interval CI, 0.90-0.99) for distinguishing early-stage HCC from cirrhosis with a sensitivity of 91% and a specificity of 90%. The AUROCs of the HCC EV ECG score remained excellent in the validation cohort (0.93; 95% CI, 0.87-0.99) and in the subgroups by etiology (viral: 0.95; 95% CI, 0.90-1.00; nonviral: 0.94; 95% CI, 0.88-0.99).
HCC EV ECG score demonstrated great potential for detecting early-stage HCC. It could augment current surveillance methods and improve patients' outcomes.
The citric acid cycle intermediate citrate plays a crucial role in metabolic processes such as fatty acid synthesis, glucose metabolism, and β-oxidation. Citrate is imported from the circulation ...across the plasma membrane into liver cells mainly by the sodium-dependent citrate transporter (NaCT; SLC13A5). Deletion of NaCT from mice led to metabolic changes similar to caloric restriction; therefore, NaCT has been proposed as an attractive therapeutic target for the treatment of obesity and type 2 diabetes. In this study, we expressed mouse and human NaCT into Xenopus oocytes and examined some basic functional properties of those transporters. Interestingly, striking differences were found between mouse and human NaCT with respect to their sensitivities to citric acid cycle intermediates as substrates for these transporters. Mouse NaCT had at least 20- to 800-fold higher affinity for these intermediates than human NaCT. Mouse NaCT is fully active at physiologic plasma levels of citrate, but its human counterpart is not. Replacement of extracellular sodium by other monovalent cations revealed that human NaCT was markedly less dependent on extracellular sodium than mouse NaCT. The low sensitivity of human NaCT for citrate raises questions about the translatability of this target from the mouse to the human situation and raises doubts about the validity of this transporter as a therapeutic target for the treatment of metabolic diseases in humans.
High LDL and/or low HDL are risk factors for atherosclerosis and are also a common clinical feature in systemic lupus erythematosus, rheumatoid arthritis, and psoriasis. Here, we show that changes in ...lipid profiles that reflect atherosclerotic disease led to activation of skin murine dendritic cells (DCs) locally, promoted dermal inflammation, and induced lymph node hypertrophy. Paradoxically, DC migration to lymph nodes was impaired, suppressing immunologic priming. Impaired migration resulted from inhibitory signals generated by platelet-activating factor (PAF) or oxidized LDL that acts as a PAF mimetic. Normal DC migration and priming was restored by HDL or HDL-associated PAF acetylhydrolase (PAFAH), which mediates inactivation of PAF and oxidized LDL. Thus, atherosclerotic changes can sequester activated DCs in the periphery where they may aggravate local inflammation even as they poorly carry out functions that require their migration to lymph nodes. In this context, HDL and PAFAH maintain a normally functional DC compartment.
OBJECTIVE—Acyl-CoA:cholesterol acyltransferase (ACAT) converts cholesterol to cholesteryl esters in plaque foam cells. Complete deficiency of macrophage ACAT has been shown to increase ...atherosclerosis in hypercholesterolemic mice because of cytotoxicity from free cholesterol accumulation, whereas we previously showed that partial ACAT inhibition by Fujirebio compound F1394 decreased early atherosclerosis development. In this report, we tested F1394 effects on preestablished, advanced lesions of apolipoprotein-E–deficient mice.
METHODS AND RESULTS—Apolipoprotein-E–deficient mice on Western diet for 14 weeks developed advanced plaques, and were either euthanized (Baseline), or continued on Western diet with or without F1394 and euthanized after 14 more weeks. F1394 was not associated with systemic toxicity. Compared with the baseline group, lesion size progressed in both groups; however, F1394 significantly retarded plaque progression and reduced plaque macrophage, free and esterified cholesterol, and tissue factor contents compared with the untreated group. Apoptosis of plaque cells was not increased, consistent with the decrease in lesional free cholesterol. There was no increase in plaque necrosis and unimpaired efferocytosis (phagocytic clearance of apoptotic cells). The effects of F1394 were independent of changes in plasma cholesterol levels.
CONCLUSION—Partial ACAT inhibition by F1394 lowered plaque cholesterol content and had other antiatherogenic effects in advanced lesions in apolipoprotein-E–deficient mice without overt systemic or plaque toxicity, suggesting the continued potential of ACAT inhibition for the clinical treatment of atherosclerosis, in spite of recent trial data.
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