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The autophagy-lysosome system is an important cellular degradation pathway that recycles dysfunctional organelles and cytotoxic protein aggregates. A decline in this system is pathogenic in many human diseases including neurodegenerative disorders, fatty liver disease, and atherosclerosis. Thus there is intense interest in discovering therapeutics aimed at stimulating the autophagy-lysosome system. Trehalose is a natural disaccharide composed of two glucose molecules linked by a ɑ-1,1-glycosidic bond with the unique ability to induce cellular macroautophagy/autophagy and with reported efficacy on mitigating several diseases where autophagy is dysfunctional. Interestingly, the mechanism by which trehalose induces autophagy is unknown. One suggested mechanism is its ability to activate TFEB (transcription factor EB), the master transcriptional regulator of autophagy-lysosomal biogenesis. Here we describe a potential mechanism involving direct trehalose action on the lysosome. We find trehalose is endocytically taken up by cells and accumulates within the endolysosomal system. This leads to a low-grade lysosomal stress with mild elevation of lysosomal pH, which acts as a potent stimulus for TFEB activation and nuclear translocation. This process appears to involve inactivation of MTORC1, a known negative regulator of TFEB which is sensitive to perturbations in lysosomal pH. Taken together, our data show the trehalose can act as a weak inhibitor of the lysosome which serves as a trigger for TFEB activation. Our work not only sheds light on trehalose action but suggests that mild alternation of lysosomal pH can be a novel method of inducing the autophagy-lysosome system. Abbreviations: ASO: antisense oligonucleotide; AU: arbitrary units; BMDM: bone marrow-derived macrophages; CLFs: crude lysosomal fractions; CTSD: cathepsin D; LAMP: lysosomal associated membrane protein; LIPA/LAL: lipase A, lysosomal acid type; MAP1LC3: microtubule-associated protein 1 light chain 3; MFI: mean fluorescence intensity; MTORC1: mechanistic target of rapamycin kinase complex 1; pMAC: peritoneal macrophages; SLC2A8/GLUT8: solute carrier family 2, (facilitated glucose transporter), member 8; TFEB: transcription factor EB; TMR: tetramethylrhodamine; TREH: trehalase

Cholesterol homeostasis has been proposed to be implicated in the development of addiction. However, the effects of ethanol on cholesterol homeostasis within the brain are not well understood. One of the most important regulators of cholesterol homeostasis is HMG-CoA reductase (HMG-CoAR), the rate-limiting enzyme of cholesterol biosynthesis. We examined the phosphorylation of HMG-CoAR and the other key regulator of lipid synthesis, acetyl-CoA carboxylase (ACC), following acute or chronic treatment with ethanol (0.5, 1, or 2 g/kg) in the rat prefrontal cortex. The phosphorylation of AMP-activated protein kinase (AMPK), which regulates the HMG-CoAR activity, and its well-known upstream regulators, was also studied. The phosphorylation of HMG-CoAR and ACC were transiently increased by ethanol treatment only in animals previously treated chronically with ethanol. Acute administration to naive animals did not induce the phosphorylation, regardless of dosage. Similarly, the phosphorylation of AMPK and the upstream regulators, LKB1 and CaMK4, were transiently increased only in chronically ethanol-treated animals. In naive animals, a high dose (2 g/kg) of ethanol decreased phosphorylation. The phosphorylation of TAK1, another upstream kinase of AMPK, was increased only from 30 min to 24 h after the chronic treatment with ethanol. Together, these results indicate that repeated exposure is required for the activating effect of ethanol on HMG-CoAR and ACC. This effect seems to be mediated by the AMPK system, and may contribute to the long-lasting neuroadaptation involved in the development of alcohol dependence.

Summary: CD137, a potent costimulatory receptor for CD8+ T cells, is expressed in various non-T cells, but little is known about its regulatory functions in these cells. In this study, we show that CD137 signaling, specifically in intestinal CD11b−CD103+ dendritic cells (DCs), restricts acute colitis progression. Mechanistically, CD137 engagement activates TAK1 and subsequently stimulates the AMPK-PGC-1α axis to enhance expression of the Aldh1a2 gene encoding the retinoic acid (RA) metabolizing enzyme RALDH2. RA can act on CD11b+CD103− DCs and induce SOCS3 expression, which, in turn, suppresses p38MAPK activation and interleukin-23 (IL-23) production. Administration of RA in DC-specific CD137−/− mice represses IL-23-producing CD11b+CD103− DCs and TH17 cells, indicating that RA is a major inhibitory effector molecule against intestinal CD11b+CD103− DCs. Additionally, the therapeutic effect of the anti-CD137 antibody is abrogated in DC-specific CD137−/− mice. Taken together, our results define a mechanism of paracrine immunoregulation operating between adjacent DC subsets in the intestine. : Jin et al. demonstrate that CD137 signaling functions as an immune checkpoint by controlling the survival and function of regulatory intestinal CD11b−CD103+ dendritic cells to coordinate the balance between regulatory T cells and pathogenic IL-23 during acute colitis. Keywords: CD137, regulatory CD11b−CD103+ DC, Foxp3+ Treg, acute colitis, retinoic acid, RALDH2, IL-23, TH17, immune checkpoint, immune tolerance

Increased oxidative stress (OS) is considered a common etiology in the pathogenesis of cardiovascular disease (CVD). Therefore, the precise regulation of reactive oxygen species (ROS) in cardiovascular cells is essential to maintain normal physiological functions. Numerous regulators of cellular homeostasis are reportedly influenced by ROS. Hydrogen peroxide (H2O2), as an endogenous ROS in aerobic cells, is a toxic substance that can induce OS. However, many studies conducted over the past two decades have provided substantial evidence that H2O2 acts as a diffusible intracellular signaling messenger. Antioxidant enzymes, including superoxide dismutases, catalase, glutathione peroxidases, and peroxiredoxins (Prdxs), maintain the balance of ROS levels against augmentation of ROS production during the pathogenesis of CVD. Especially, Prdxs are regulatory sensors of transduced intracellular signals. The intracellular abundance of Prdxs that specifically react with H2O2 act as regulatory proteins. In this review, we focus on the role of Prdxs in the regulation of ROS-induced pathological changes in the development of CVD.

High protein diets are commonly utilized for weight loss, yet have been reported to raise cardiovascular risk. The mechanisms underlying this risk are unknown. Here, we show that dietary protein drives atherosclerosis and lesion complexity. Protein ingestion acutely elevates amino acid levels in blood and atherosclerotic plaques, stimulating macrophage mTOR signaling. This is causal in plaque progression as the effects of dietary protein are abrogated in macrophage-specific Raptor-null mice. Mechanistically, we find amino acids exacerbate macrophage apoptosis induced by atherogenic lipids, a process that involves mTORC1-dependent inhibition of mitophagy, accumulation of dysfunctional mitochondria, and mitochondrial apoptosis. Using macrophage-specific mTORC1- and autophagy-deficient mice we confirm this amino acid-mTORC1-autophagy signaling axis in vivo. Our data provide the first insights into the deleterious impact of excessive protein ingestion on macrophages and atherosclerotic progression. Incorporation of these concepts in clinical studies will be important to define the vascular effects of protein-based weight loss regimens.

Oxidative stress activates macroautophagy/autophagy and contributes to atherogenesis via lipophagic flux, a form of lipid removal by autophagy. However, it is not known exactly how endogenous antioxidant enzymes are involved in lipophagic flux. Here, we demonstrate that the antioxidant PRDX1 (peroxiredoxin 1) has a crucial role in the maintenance of lipophagic flux in macrophages. PRDX1 is more highly expressed than other antioxidant enzymes in monocytes and macrophages. We determined that Prdx1 deficiency induced excessive oxidative stress and impaired maintenance of autophagic flux in macrophages. Prdx1-deficient macrophages had higher intracellular cholesterol mass and lower cholesterol efflux compared with wild type. This perturbation in cholesterol homeostasis was due to impaired lipophagic cholesterol hydrolysis caused by excessive oxidative stress, resulting in the inhibition of free cholesterol formation and the reduction of NR1H3 (nuclear receptor subfamily 1, group H, member 3) activity. Notably, impairment of both lipophagic flux and cholesterol efflux was restored by the 2-Cys PRDX-mimics ebselen and gliotoxin. Consistent with this observation, apoe −/− mice transplanted with bone marrow from prdx1−/−apoe−/− mice had increased plaque formation compared with apoe−/− BM-transplanted recipients. This study reveals that PRDX1 is crucial to regulating lipophagic flux and maintaining macrophage cholesterol homeostasis against oxidative stress. We suggest that PRDX1-dependent control of oxidative stress may provide a strategy for treating atherosclerosis and autophagy-related human diseases.

Macrophage cholesterol efflux is a central step in reverse cholesterol transport, which helps to maintain cholesterol homeostasis and to reduce atherosclerosis. Lipophagy has recently been identified as a new step in cholesterol ester hydrolysis that regulates cholesterol efflux, since it mobilizes cholesterol from lipid droplets of macrophages via autophagy and lysosomes. In this review, we briefly discuss recent advances regarding the mechanisms of the cholesterol efflux pathway in macrophage foam cells, and present lipophagy as a therapeutic target in the treatment of atherosclerosis.

In the atherosclerotic plaque, macrophages are the key catabolic workhorse responsible for clearing lipid and dead cell debris. To survive the highly proinflammatory and lipotoxic plaque environment, macrophages must adopt strategies for maintaining tight homeostasis and self-renewal. Macroautophagy/autophagy is a pro-survival cellular pathway wherein damaged or excess cellular cargoes are encapsulated by a double-membrane compartment and delivered to the lysosome for hydrolysis. Previously, macrophage-specific autophagy deficiency has been shown to be atherogenic through several complementary mechanisms including hyperactivation of the inflammasome, defective efferocytosis, accumulation of cytotoxic protein aggregates, and impaired lipid degradation. Conversely, in a recent study we hypothesized that enhancing the macrophage autophagy-lysosomal system through genetic or pharmacological means could protect against atherosclerosis. We demonstrated that TFEB, a transcription factor master regulator of autophagy and lysosome biogenesis, coordinately enhances the function of this system to reduce atherosclerotic plaque burden. Further, we characterized the disaccharide trehalose as a novel inducer of TFEB with similar atheroprotective effects. Overall, these findings mechanistically interrogate the importance and therapeutic promise of a functional autophagy-lysosome degradation system in plaque macrophage biology.

TFEB is a basic helix-loop-helix transcription factor that confers protection against metabolic diseases such as atherosclerosis by targeting a network of genes involved in autophagy-lysosomal biogenesis and lipid catabolism. In this study, we sought to characterize the role of TFEB in adipocyte and adipose tissue physiology and evaluate the therapeutic potential of adipocyte-specific TFEB overexpression in obesity. We demonstrated that mice with adipocyte-specific TFEB overexpression (Adipo-TFEB) were protected from diet-induced obesity, insulin resistance, and metabolic sequelae. Adipo-TFEB mice were lean primarily through increased metabolic rate, suggesting a role for adipose tissue browning and enhanced nonshivering thermogenesis in fat. Transcriptional characterization revealed that TFEB targeted genes involved in adipose tissue browning rather than those involved in autophagy. One such gene encoded PGC-1α, an established target of TFEB that promotes adipocyte browning. To dissect the role of PGC-1α in mediating the downstream effects of TFEB overexpression, we generated mice with adipocyte-specific PGC-1α deficiency and TFEB overexpression. Without PGC-1α, the ability of TFEB overexpression to brown adipose tissue and to elicit beneficial metabolic effects was blunted. Overall, these data implicate TFEB as a PGC-1α-dependent regulator of adipocyte browning and suggest its therapeutic potential in treating metabolic disease.

The present study aimed to investigate the influence of the host retinal microenvironment on cell migration and differentiation using Neuro2a (N2a) cells transduced with green fluorescent protein. N2a cells were transplanted into the vitreous cavities of developing mouse eyes (C57BL/6) on postnatal days 1, 5, and 10 (P1, 5, and 10). To analyze the effects of the host microenvironment on neural differentiation of N2a cells in vitro, cells were treated with a conditioned medium (CM) collected from retinal cells cultured at each developmental stage. We observed that numerous cells transplanted into P5 mice eyes migrated into all layers of the host retina, and the presence of processes indicated morphological differentiation. Some transplanted N2a cells expressed several neural markers. However, cells transplanted into the P1 and 10 mice eyes only proliferated within the vitreous cavity. Neurite length increased in N2a cells treated with CM collected from the cultured retinal cells from P5 and 10 mice, while western blotting revealed that the levels of proteins related to neural differentiation were not significantly altered in N2a cells treated with CM. We show that the migration and differentiation capacities of transplanted cells were differentially influenced by the microenvironment of the retinal postnatal ontogeny.

The compounds 1,8-cineole and zerumbone (ZER) from the Cyperus rotundus rhizome along with another 11 previously identified rhizome essential oil constituents and α-humulene, which lacks the only carbonyl group present in ZER, as well as binary mixtures of ZER and seven active compounds were tested for repellency to male B. germanica. The results were compared to N,N-diethyl-3-methylbenzamide (deet). In filter-paper choice tests, ZER was the most repellent compound, and α-humulene was ineffective, which indicates that the α,β-unsaturated carbonyl group of ZER is a prerequisite component for repellency. At 81.5 μg cm−2, enhanced repellency was produced by binary mixtures of ZER and 1,8-cineole, (+)-dihydrocarvone or (R)-(+)-limonene (70:30, 50:50 and 30:70 ratios by weight). These mixtures were very effective against male B. germanica within 24 h and were more repellent than a single compound or deet alone. The optimum ZER content was determined to be more than 50%. In Ebeling choice box tests at 652.4 μg cm−2, these compounds and deet resulted in complete repellency to intact male B. germanica, while they exhibited 35–47% repellency to antennectomized male one. Mixtures formulated from the active constituents of the C. rotundus rhizome could be useful as potential repellents for controlling B. germanica.

CD137 (4-1BB), a member of the tumor necrosis factor receptor superfamily, has been reported to be expressed in atherosclerotic plaques, and to promote lesion formation. However, the role of CD137 in mediating atherosclerotic plaque stability and the possible underlying molecular and cellular mechanisms are poorly understood. Here, apolipoprotein E-deficient (ApoE(-/-)) and CD137-deficient ApoE(-/-) (ApoE(-/-)CD137(-/-)) mice fed a chow diet for 66 wk were used. CD137 induces plaque instability, which is characterized by increased plaque necrosis, decreased collagen content, decreased vascular smooth muscle cell (VSMC) content, and increased macrophage infiltration. CD137 also increases the infiltration of effector T (Teff) cells into plaque lesion sites, resulting in increased interferon-γ (IFN-γ) expression. Interestingly, Teff-cell-derived IFN-γ inhibits collagen synthesis in atherosclerotic plaques. Furthermore, CD137 activation increases the apoptosis of VSMCs, possibly by decreasing the antiapoptotic regulator, Bcl-2, and subsequently up-regulating cleaved caspase-3. In macrophages, activation of CD137 signaling boosted the oxidized low density lipoprotein-induced expression of matrix metalloproteinase 9 via the p38 mitogen-activated protein kinase and extracellular signal-regulated kinase1/2 signaling pathways. In summary, activation of CD137 signaling decreases the stability of advanced atherosclerotic plaques via its combined effects on Teff cells, VSMCs, and macrophages.

In this paper, fabricated by MEMS uncooled micro-bolometer detector for the study in the infrared sensitivity enhancement. Absorption layer SiOx-Metal series MDTF (metal-dielectric thin film) by high absorption rate and has a high thermal coefficient of resistance, low noise characteristics were implemented. Then MDTF were made in a vacuum deposition method. And MDTF for the analysis of the physical properties of silicon wafers were fabricated, TCR (temperature coefficient of resistance) value was made in order to measure the glass wafer and FT-IR (Fourier Transform Infrared spectroscopy) values were made in order to measure the germanium window. The analyzed results of MDTF -3 [%/K] has more characteristics of the TCR. And 8~12 um wavelength region close to 70% in the absorption characteristic.

In this study, the synergistic effect of 6-[4-(1-cyclohexyl- 1H-tetrazol-5-yl) butoxy]-3,4-dihydro-2(1H )-quinolinone (cilostazol) and Ginkgo biloba extract (GbE) was examined in apolipoprotein E (ApoE) null mice. Co-treatment with GbE and cilostazol synergistically decreased reactive oxygen species (ROS) production in ApoE null mice fed a high-fat diet. Co-treatment resulted in a significantly decreased atherosclerotic lesion area compared to untreated ApoE mice. The inflammatory cytokines and adhesion molecules such as monocyte chemoattractant-1 (MCP-1), soluble vascular cell adhesion molecule-1 (sVCAM-1), and VCAM-1 which can initiate atherosclerosis were significantly reduced by the co-treatment of cilostazol with GbE. Further, the infiltration of macrophages into the intima was decreased by co-treatment. These results suggest that co-treatment of GbE with cilostazol has a more potent anti-atherosclerotic effect than treatment with cilostazol alone in hyperlipidemic ApoE null mice and could be a valuable therapeutic strategy for the treatment of atherosclerosis.

In the present study, we have investigated the effect of fisetin on the apoptosis and DNA single strand breaks in ultraviolet light B (UVB)-exposed NIH3T3 cells. Exposure of cells to UVB light and post-incubation in growth medium for 48 hr resulted in about 50% of cells with apoptotic nuclear fragmentation. Addition of various concentrations of fisetin in the postincubation medium, however, significantly reduced the apoptotic nuclear fragmentation as compared with the values expected when the effects are additive and independent. DNA single strand breaks induced by UVB exposure were also significantly decreased by postincubation with fisetin. By Western blot analysis, fisetin post-incubation was shown to attenuate the p53 upregulation upon UVB exposure. Furthermore, the decrease of proliferating cell nuclear antigen (PCNA) level upon UVB exposure was alleviated by fisetin postincubation. These results suggest that fisetin decrease the apoptosis and increae DNA repair in a possible association with alteration of p53 and PCNA levels in UVB-exposed cells.

Rhus verniciflua stokes (RVS) is a popular medicinal plant in oriental medicines which is commonly used to resolve extravasated blood. To elucidate the molecular mechanism of the role of RVS extracts on the regulation of lipid and cholesterol biosynthesis, we investigated whether RVS extract protect the hyperlipidemia in western diet-induced C57BL6/J mice. Mice fed a western diet and additionally RVS extracts was administered orally at a dose of 0.1 or 1 g/kg/day for 2 weeks respectively. Group with higher dose of RVS extract showed a significantly decreased body weight compared with western diet fed mice groups. And total cholesterol, LDL-cholesterol levels and fatty liver formation were also improved especially in group of mice fed western diet supplemented high dose RVS extracts. Next, synthesis of hepatic bile acids were significantly increased in RVS extract fed groups. Furthermore, RVS extracts significantly increase promoter activity of Cyp7a1 via up-regulate the transcriptional expression level of LXRα. Our data suggest that RVS extracts could be a potent therapeutic ingredient for prevent a hyperlipidemia via increase of bile acids biosynthesis.

Hyperlipidemia is a well-recognized risk factor for atherosclerosis and can be regulated by adipokines. Expression of the adipokine resistin-like molecule alpha (Retnla) is regulated by food intake; whether Retnla has a role in the pathogenesis of hyperlipidemia and atherosclerosis is unknown. Here we report that Retnla has a cholesterol-lowering effect and protects against atherosclerosis in low-density lipoprotein receptor-deficient mice. On a high-fat diet, Retnla deficiency promotes hypercholesterolaemia and atherosclerosis, whereas Retnla overexpression reverses these effects and improves the serum lipoprotein profile, with decreased cholesterol in the very low-density lipoprotein fraction concomitant with reduced serum apolipoprotein B levels. We show that Retnla upregulates cholesterol-7-alpha-hydroxylase, a key hepatic enzyme in the cholesterol catabolic pathway, through induction of its transcriptional activator liver receptor homologue-1, leading to increased excretion of cholesterol in the form of bile acids. These findings define Retnla as a novel therapeutic target for treating hypercholesterolaemia and atherosclerosis.

Rationale: Peroxiredoxin 2 (Prdx2), a thiol-specific peroxidase, has been reported to regulate proinflammatory responses, vascular remodeling, and global oxidative stress. Objective: Although Prdx2 has been proposed to retard atherosclerosis development, no direct evidence and mechanisms have been reported. Methods and Results: We show that Prdx2 is highly expressed in endothelial and immune cells in atherosclerotic lesions and blocked the increase of endogenous H 2 O 2 by atherogenic stimulation. Deficiency of Prdx2 in apolipoprotein E–deficient (ApoE −/− ) mice accelerated plaque formation with enhanced activation of p65, c-Jun, JNKs, and p38 mitogen-activated protein kinase; and these proatherogenic effects of Prdx2 deficiency were rescued by administration of the antioxidant ebselen. In bone marrow transplantation experiments, we found that Prdx2 has a major role in inhibiting atherogenic responses in both vascular and immune cells. Prdx2 deficiency resulted in increased expression of vascular adhesion molecule-1, intercellular adhesion molecule-1, and monocyte chemotactic protein-1, which led to increased immune cell adhesion and infiltration into the aortic intima. Compared with deficiency of glutathione peroxidase 1 or catalase, Prdx2 deficiency showed a severe predisposition to develop atherosclerosis. Conclusions: Prdx2 is a specific peroxidase that inhibits atherogenic responses in vascular and inflammatory cells, and specific activation of Prdx2 may be an effective means of antiatherogenic therapy.

Ginseng has been used worldwidely as a traditional medicine of Asian countries for treatment of various diseases including cancer. The purpose of this study was to determine the effect of ginseng saponin mRg2, a mixture of ginsenosides containing 60% Rg2, on the repair and apoptosis of ultraviolet B (UVB)-exposed NIH3T3 cells. When cells were exposed to UVB and then incubated with normal growth medium for 48 h, cell viability, as determined by trypan blue exclusion assay decreased to about 25%. However, when mRg2 was included in the postincubation medium, the UVB-induced loss of cell viability was significantly reduced as compared with that postincubated in normal growth medium. 4,6-diamidino-2-phenylindole (DAPI) staining showed that postincubation of the UVB-exposed cells in medium containing mRg2 significantly reduced the apoptotic nuclear fragmentation. Interestingly, when cells were preincubated with mRg2 for 24 h and then exposed to various doses of UV, the amount of repair synthesis significantly increased as compared with those in cells exposed to UVB alone. Western blot analysis indicated that the mRg2 postincubation after UVB exposure potentiated the level of p53 and p21. The level of Triton nonextractable proliferating cell nuclear antigen (PCNA) also remained elevated by mRg2 postincubation. All these results suggest that mRg2 protects cells against UVB-induced genotoxicity by increasing DNA repair and decreasing apoptosis, in possible association with the modulation of protein levels involved in cell cycle arrest or progression.

In this study we investigated the effect of ginseng saponins on the p53-dependent apoptosis in NIH3T3 cells exposed to methyl methanesulfonate (MMS), an alkylating agent. Trypan blue exclusion assay, cell morphology studies, and apoptotic index determined by acridine orange staining showed that the postincubation of MMS-exposed cells in medium containing diol- (PD) or triol-type (PT) ginseng saponins potentiate the apoptotic cell death. FACS analysis indicated that the increased apoptotic cell population in the saponin-postincubation group was accompanied by the accumulation of cells in G0/G1 phase. By Western blot analyses it was demonstrated that postincubation of saponins increases the expression of p53 and p21 in MMS-exposed cells but decreased that of CDK2, cyclin E and D1, and PCNA. The upregulation of p53 and p21 and downregulation of CDK2 was shown to be p53-dependent in experiments using the p53 antisense oligonucleotide. These results suggest that ginseng saponins contain components potentiating the apoptosis of MMS-exposed NIH3T3 cells via p53 and p21 activation, accompanied with by downregulation of cell cycle-related protein expression.