The 100 most-cited articles in The FASEB Journal, 2016

From yeast to mammals, autophagy is an important mechanism for sustaining cellular homeostasis through facilitating the degradation and recycling of aged and cytotoxic components. During autophagy, cargo is captured in double-membraned vesicles, the autophagosomes, and degraded through lysosomal fusion. In yeast, autophagy initiation, cargo recognition, cargo engulfment, and vesicle closure is Atg8 dependent. In higher eukaryotes, Atg8 has evolved into the LC3/GABARAP protein family, consisting of 7 family proteins [LC3A (2 splice variants), LC3B, LC3C, GABARAP, GABARAPL1, and GABARAPL2]. LC3B, the most studied family protein, is associated with autophagosome development and maturation and is used to monitor autophagic activity. Given the high homology, the other LC3/GABARAP family proteins are often presumed to fulfill similar functions. Nevertheless, substantial evidence shows that the LC3/GABARAP family proteins are unique in function and important in autophagy-independent mechanisms. In this review, we discuss the current knowledge and functions of the LC3/GABARAP family…

Efficient intercellular transfer of RNAs, proteins, and lipids as protected exosomal cargo has been demonstrated in the CNS, but distinct physiologic and pathologic roles have not been well defined for this pathway. The capacity to isolate immunochemically human plasma neuron-derived exosomes (NDEs), containing neuron-specific cargo, has permitted characterization of CNS-derived exosomes in living humans. Constituents of the amyloid β-peptide (Aβ)42-generating system now are examined in 2 distinct sets of human neural cells by quantification in astrocyte-derived exosomes (ADEs) and NDEs, enriched separately from plasmas of patients with Alzheimer's disease (AD) or frontotemporal dementia (FTD) and matched cognitively normal controls. ADE levels of β-site amyloid precursor protein-cleaving enzyme 1 (BACE-1), γ-secretase, soluble Aβ42, soluble amyloid precursor protein (sAPP)β, sAPPα, glial-derived neurotrophic factor (GDNF), P-T181-tau, and P-S396-tau were significantly (3- to 20-fold) higher than levels in NDEs for patients and controls. BACE-1 levels also were a mean of 7-fold…

Synaptic dysfunction occurs early in senile dementias, presumably as a result of decreased levels of functional synaptic proteins as found in autopsied brains of patients with Alzheimer's disease (AD) or frontotemporal dementia (FTD). Plasma neuronal-derived exosomes (NDEs) were recovered by precipitation and immunoabsorption from 12 patients with AD, 16 with FTD, and 28 controls in a cross-sectional study, and from 9 patients with AD, 10 with FTD, and 19 controls in a longitudinal study. Six synaptic proteins in NDE extracts were quantified by ELISAs and normalized for exosome amounts. NDE levels of synaptophysin, synaptopodin, synaptotagmin-2, and neurogranin were significantly lower in patients with FTD and AD than in controls, but those of growth-associated protein 43 and synapsin 1 were reduced only in patients with AD. Functionally relevant phosphorylation of synapsin 1 serine 9 was reduced in patients with FTD and AD, although total synapsin 1 protein was higher in FTD than in controls. NDE levels of synaptotagmin, synaptophysin, and neurogranin were decreased years before…

In June 2015, the National Institutes of Health (NIH) released a Guide notice (NOT-OD-15-102) that highlighted the expectation of the NIH that the possible role of sex as a biologic variable be factored into research design, analyses, and reporting of vertebrate animal and human studies. Anticipating these guidelines, the NIH Office of Research on Women's Health, in October 2014, convened key stakeholders to discuss methods and techniques for integrating sex as a biologic variable in preclinical research. The workshop focused on practical methods, experimental design, and approaches to statistical analyses in the use of both male and female animals, cells, and tissues in preclinical research. Workshop participants also considered gender as a modifier of biology. This article builds on the workshop and is meant as a guide to preclinical investigators as they consider methods and techniques for inclusion of both sexes in preclinical research and is not intended to prescribe exhaustive/specific approaches for compliance with the new NIH policy.-Miller, L. R., Marks, C., Becker, J. B.,…

Recent studies have emphasized the role of perivascular inflammation in cardiovascular disease. We studied mechanisms of perivascular leukocyte infiltration in angiotensin II (Ang II)-induced hypertension and their links to vascular dysfunction. Chronic Ang II infusion in mice increased immune cell content of T cells (255 ± 130 to 1664 ± 349 cells/mg; P < 0.01), M1 and M2 macrophages, and dendritic cells in perivascular adipose tissue. In particular, the content of T lymphocytes bearing CC chemokine receptor (CCR) 1, CCR3, and CCR5 receptors for RANTES chemokine was increased by Ang II (CCR1, 15.6 ± 1.5% vs. 31 ± 5%; P < 0.01). Hypertension was associated with an increase in perivascular adipose tissue expression of the chemokine RANTES (relative quantification, 1.2 ± 0.2 vs. 3.5 ± 1.1; P < 0.05), which induced T-cell chemotaxis and vascular accumulation of T cells expressing the chemokine receptors CCR1, CCR3, and CCR5. Mechanistically, RANTES(-/-) knockout protected against vascular leukocyte, and in particular T lymphocyte infiltration (26 ± 5% in wild type Ang II vs. 15 ± 4% in…

Epidemiologic evidence suggests that air pollution is a risk factor for childhood obesity. Limited experimental data have shown that early-life exposure to ambient particles either increases susceptibility to diet-induced weight gain in adulthood or increases insulin resistance, adiposity, and inflammation. However, no data have directly supported a link between air pollution and non-diet-induced weight increases. In a rodent model, we found that breathing Beijing's highly polluted air resulted in weight gain and cardiorespiratory and metabolic dysfunction. Compared to those exposed to filtered air, pregnant rats exposed to unfiltered Beijing air were significantly heavier at the end of pregnancy. At 8 wk old, the offspring prenatally and postnatally exposed to unfiltered air were significantly heavier than those exposed to filtered air. In both rat dams and their offspring, after continuous exposure to unfiltered air we observed pronounced histologic evidence for both perivascular and peribronchial inflammation in the lungs, increased tissue and systemic oxidative stress,…

Bile acids are synthesized from cholesterol and are known to be involved with the emulsification and digestion of dietary lipids and fat-soluble vitamins. Outside of this role, bile acids can act as cell signaling effectors through binding and activating receptors on both the cell membrane and nucleus. Numerous reports have investigated these signaling pathways in conditions where the liver is damaged. More recently, effort has been made to investigate the role of bile acids in diseases outside of those associated with liver damage. This review summarizes recent findings on the influences that bile acids can exert in normal neurological function and their contribution to diseases of the nervous system, with the intent of highlighting the role of these metabolites as potential players in neurological disorders.-McMillin, M., DeMorrow, S. Effects of bile acids on neurological function and disease.

Epigenetics plays an important role in orchestrating key biologic processes. Epigenetic marks, including DNA methylation, histones, chromatin structure, and noncoding RNAs, are modified throughout life in response to environmental and behavioral influences. With each new generation, DNA methylation patterns are erased in gametes and reset after fertilization, probably to prevent these epigenetic marks from being transferred from parents to their offspring. However, some recent animal studies suggest an apparent resistance to complete erasure of epigenetic marks during early development, enabling transgenerational epigenetic inheritance. Whether there are similar mechanisms in humans remains unclear, with the exception of epigenetic imprinting. Nevertheless, a distinctly different mechanism-namely, intrauterine exposure to environmental stressors that may affect establishment of the newly composing epigenetic patterns after fertilization-is often confused with transgenerational epigenetic inheritance. In this review, we delineate the definition of and requirement for…

Air pollution is implicated in neurodegenerative disease risk and progression and in microglial activation, but the mechanisms are unknown. In this study, microglia remained activated 24 h after ozone (O3) exposure in rats, suggesting a persistent signal from lung to brain. Ex vivo analysis of serum from O3-treated rats revealed an augmented microglial proinflammatory response and β-amyloid 42 (Aβ42) neurotoxicity independent of traditional circulating cytokines, where macrophage-1 antigen-mediated microglia proinflammatory priming. Aged mice exhibited reduced pulmonary immune profiles and the most pronounced neuroinflammation and microglial activation in response to mixed vehicle emissions. Consistent with this premise, cluster of differentiation 36 (CD36)(-/-) mice exhibited impaired pulmonary immune responses concurrent with augmented neuroinflammation and microglial activation in response to O3 Further, aging glia were more sensitive to the proinflammatory effects of O3 serum. Together, these findings outline the lung-brain axis, where air pollutant exposures result in…

Increasing energy expenditure through activation of brown adipose tissue (BAT) is a critical approach to treating obesity and diabetes. In this study, rutin, a natural compound extracted from mulberry and a drug used as a capillary stabilizer clinically for many years without any side effects, regulated whole-body energy metabolism by enhancing BAT activity. Rutin treatment significantly reduced adiposity, increased energy expenditure, and improved glucose homeostasis in both genetically obese (Db/Db) and diet-induced obesity (DIO) mice. Rutin also induced brown-like adipocyte (beige) formation in subcutaneous adipose tissue in both obesity mouse models. Mechanistically, we found that rutin directly bound to and stabilized SIRT1, leading to hypoacetylation of peroxisome proliferator-activated receptor γ coactivator-1α protein, which stimulated Tfam transactivation and eventually augmented the number of mitochondria and UCP1 activity in BAT. These findings reveal that rutin is a novel small molecule that activates BAT and may provide a novel therapeutic approach to the treatment of…

Long noncoding RNAs (lncRNAs) are emerging as important regulators in cellular processes, including the development, proliferation, and migration of cancer cells. We have demonstrated in a prior study that small nucleolar RNA host gene 5 (SNHG5) is dysregulated in gastric cancer (GC). To further explore the underlying mechanisms of SNGH5 function in the development of GC, in this study, we screened the microRNAs interacting with SNHG5 and elucidated their roles in GC. We showed that SNHG5 contains a putative miR-32-binding site and that deletion of this site abolishes the responsiveness to miR-32. Suppression of SNHG5 expression by miR-32 was found to be Argonaute (Ago)2-dependent. Immunoprecipitation showed that SNHG5 could be pulled down from the Ago-2 complex with miR-32. Furthermore, it was reported that Kruppel-like factor 4 (KLF4) is a target gene of miR-32. In agreement with SNHG5 being a decoy for miR-32, we showed that KLF4 suppression by miR-32 could be partially rescued by SNHG5 overexpression, whereas miR-32 mimic rescued SNHG5 overexpression-mediated suppression of GC…

HIV-infected individuals have activated monocytes with an IFNα phenotype and elevated levels of circulating LPS. These individuals also have a risk of premature cardiovascular disease. The effect of activated monocyte exosomes (Exos) on endothelial cells is unknown. To determine whether Exos from immune-activated monocytes could alter endothelial cell expression and contribute to monocyte/macrophage transmigration and adhesion, we isolated Exos from monocytes stimulated with IFNα, LPS, or both (I/L). We show that monocyte Exos contain different inflammatory microRNA cargo depending on stimulation. When LPS Exos or I/L Exos were added to HUVECs, we found a significant increase in adhesion molecule ICAM-1, chemokine ligand (CCL)-2, and cytokine IL-6 mRNAs and proteins compared with cells treated with IFNα Exos or Exos derived from unstimulated monocytes. Inhibition of transcription factor NF-κB, a common inflammatory cytokine pathway, prevented induction of CCL2, IL6, and ICAM1 Inhibition of TLR4 resulted in differential blockage of the targets. Our results demonstrate for the first…

ABSTRACT Ketone bodies are the most energy‐efficient fuel and yield more ATP permole of substrate than pyruvate and increase the free energy released from ATP hydrolysis. Elevation of circulating ketones via high‐fat, low carbohydrate diets has been used for the treatment of drug‐refractory epilepsy and for neuro degenerative diseases, such as Parkinson's disease. Ketones may also be beneficial for muscle and brain in times of stress, such as endurance exercise. The challenge has been to raise circulating ketone levels by using a palatable diet without altering lipid levels. We found that blood ketone levels can be increased and cholesterol and triglycerides decreased by feeding rats a novel ketone ester diet: chow that is supplemented with ( R )‐3‐hydroxybutyl ( R )‐3‐hydroxybutyrate as 30% of calories. For 5 d, rats on the ketone diet ran 32% further on a tread mill than did control rats that ate an isocaloric diet that was supplemented with either corn starch or palmoil ( P < 0.05). Ketone‐fed rats completed an 8‐ arm radial maze test 38% faster than did those on the other diets,…

Medium-chain triglycerides have been used as part of a ketogenic diet effective in reducing epileptic episodes. The health benefits of the derived medium-chain fatty acids (MCFAs) are thought to result from the stimulation of liver ketogenesis providing fuel for the brain. We tested whether MCFAs have direct effects on energy metabolism in induced pluripotent stem cell-derived human astrocytes and neurons. Using single-cell imaging, we observed an acute pronounced reduction of the mitochondrial electrical potential and a concomitant drop of the NAD(P)H signal in astrocytes, but not in neurons. Despite the observed effects on mitochondrial function, MCFAs did not lower intracellular ATP levels or activate the energy sensor AMP-activated protein kinase. ATP concentrations in astrocytes were unaltered, even when blocking the respiratory chain, suggesting compensation through accelerated glycolysis. The MCFA decanoic acid (300 μM) promoted glycolysis and augmented lactate formation by 49.6%. The shorter fatty acid octanoic acid (300 μM) did not affect glycolysis but increased the rates…

ABSTRACT Inflammatory factors secreted by microglia play an important role in focal ischemic stroke. The mammalian target of rapamycin (mTOR) pathway is a known regulator of immune responses, but the role that mTORC1 signaling plays in poststroke neuroinflammation is not clear. To explore the relationship between microglial action in the mTORC1 pathway and the impact on stroke, we administered the mTORC1 inhibitors sirolimus and everolimus to mice. Presumably, disrupting the mTORC1 pathway after focal ischemic stroke should clarify the subsequent activity of microglia. For that purpose, we generated mice deficient in the regulatory associated protein of mTOR ( Raptor ) in microglia, whose mTORC1 signaling was blocked, by crossing Raptor loxed ( Raptor flox/flox ) mice with CX3CR1 CreER mice, which express Cre recombinase under the control of the CX3C chemokine receptor 1 promoter. mTORC1 blockade reduced lesion size, improved motor function, dramatically decreased production of pro‐inflammatory cytokines and chemokines, and reduced the number of M1 type microglia. Thus, mTORC1…

ABSTRACT Altered energy balance and insulin resistance are important characteristics of aging. Skeletal muscle is a major site of glucose disposal, and the role of aging‐associated inflammation in skeletal muscle insulin resistance remains unclear. To investigate, we examined glucose metabolism in 18‐mo‐old transgenic mice with muscle‐specific overexpression of IL‐10 (M IL10 ) and in wild‐type mice during hyperinsulinemic‐euglycemic clamping. Despite similar fat mass and energy balance, M IL10 mice were protected from aging‐associated insulin resistance with significant increases in glucose infusion rates, whole‐body glucose turnover, and skeletal muscle glucose uptake (~60%; P <0.05), as compared to age‐matched WT mice. This protective effect was associated with decreased muscle inflammation, but no changes in adipose tissue inflammation in aging M IL10 mice. These results demonstrate the importance of skeletal muscle inflammation in aging‐mediated insulin resistance, and our findings further implicate a potential therapeutic role of anti‐inflammatory cytokine in the treatment of…

Chicoric acid (CA), a natural phenolic acid extracted from chicory and the echinacea (purple coneflower) plant ( Echinacea purpurea ), has been regarded as a nutraceutical that has powerful antioxidant and antiobesity activities. We investigated the inhibitory effects of CA on systemic inflammation‐induced neuroinflammation, amyloidogenesis, and cognitive impairment. C57BL/6J mice were treated with 0.05% CA in the drinking water for 45 d. The mice were then treated by intraperitoneal injection of lipopolysaccharide (LPS). It was found that CA prevented LPS‐induced memory impairment and neuronal loss through behavioral tests and histological exam‐ination. Furthermore, amyloidogenesis in the CNS was detected. The results showed that CA prevented LPS‐induced increases in amyloid β (1–42 specific) (Aβ 1–42 ) accumulation, levels of amyloid precursor protein, and neuronal β‐secretase 1 (BACE1), as well as the equilibrium cholinergic system in mouse brain. Moreover, CA down‐regulated LPS‐induced glial overactivation by inhibiting the MAPK and NF‐κB pathway. Consequently, CA reduced the…

Inflammation in arterial walls leads to coronary artery disease (CAD). Because specialized proresolving lipid mediators (SPMs; lipoxins, resolvins, and protectins) stimulate resolution of inflammation in animal models, we tested whether n-3 fatty acids impact SPM profiles in patients with CAD and promote clot remodeling. Six patients with stable CAD were randomly assigned to either treatment with daily 3.36 g Lovaza for 1 yr or without. Targeted lipid mediator-metabololipidomics showed that both groups had absence of resolvin D1 (RvD1), RvD2, RvD3, RvD5 and resolvin E1-all of which are present in healthy patients. Those not taking Lovaza had an absence of aspirin-triggered resolvin D3 (AT-RvD3) and aspirin-triggered lipoxin B4 (AT-LXB4). Lovaza treatment restored AT-RvD3 and AT-LXB4 and gave levels of RvD6 and aspirin-triggered protectin D1 (AT-PD1) twice as high (resolvin E2 ∼5 fold) as well as lower prostaglandins. Principal component analysis indicated positive relationships for patients with CAD who were receiving Lovaza with increased AT-RvD3, RvD6, AT-PD1, and AT-LXB4 SPMs…

Increased mitochondrial content and respiration have both been reported after exercise training. However, no study has directly compared how different training volumes influence mitochondrial respiration and markers of mitochondrial biogenesis. Ten healthy men performed high-intensity interval cycling during 3 consecutive training phases; 4 wk of normal-volume training (NVT; 3/wk), followed by 20 d of high-volume training (HVT; 2/d) and 2 wk of reduced-volume training (RVT; 5 sessions). Resting biopsy samples (vastus lateralis) were obtained at baseline and after each phase. No mitochondrial parameter changed after NVT. After HVT, mitochondrial respiration and citrate synthase activity (∼40-50%), as well as the protein content of electron transport system (ETS) subunits (∼10-40%), and that of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), NRF1, mitochondrial transcription factor A (TFAM), PHF20, and p53 (∼65-170%) all increased compared to baseline; mitochondrial specific respiration remained unchanged. After RVT, all the mitochondrial parameters measured…

Impairment of amyloid-β (Aβ) clearance leads to Aβ accumulation in the brain during the development of Alzheimer's disease (AD). Strategies that can restore or improve the clearance function hold great promise in delaying or preventing the onset of AD. Here, we show that n-3 polyunsaturated fatty acids (PUFAs), by use of fat-1 transgenic mice and oral administration of fish oil, significantly promote interstitial Aβ clearance from the brain and resist Aβ injury. Such beneficial effects were abolished in Aqp4-knockout mice, suggesting that the AQP4-dependent glymphatic system is actively involved in the promoting the effects of n-3 PUFAs on the clearance of extracellular Aβ. Imaging on clarified brain tissues clearly displayed that n-3 PUFAs markedly inhibit the activation of astrocytes and protect the AQP4 polarization in the affected brain region after Aβ injection. The results of the present study prove a novel mechanism by which n-3 PUFAs exert protective roles in reducing Aβ accumulation via mediating the glymphatic system function.-Ren, H., Luo, C., Feng, Y., Yao, X., Shi, Z.,…

The Wnt/β-catenin pathway is an evolutionarily conserved set of signals with critical roles in embryonic and neonatal development across species. In mammals the pathway is quiescent in many organs. It is reactivated in response to injury and is reported to play complex and contrasting roles in promoting regeneration and fibrosis. We review the current understanding of the role of the Wnt/β-catenin pathway in injury of various mammalian organs and discuss the current advances and potential of Wnt inhibitory therapeutics toward promoting tissue regeneration and reducing fibrosis.-Bastakoty, D., Young, P. P. Wnt/β-catenin pathway in tissue injury: roles in pathology and therapeutic opportunities for regeneration.

Obesity is an epidemic, complex disease that is characterized by increased glucose, lipids, and low-grade inflammation in the circulation, among other factors. It creates the perfect scenario for the production of ceramide, the building block of the sphingolipid family of lipids, which is involved in metabolic disorders such as obesity, diabetes, and cardiovascular disease. In addition, obesity causes a decrease in fatty acid oxidation (FAO), which contributes to lipid accumulation within the cells, conferring more susceptibility to cell dysfunction. C16:0 ceramide, a specific ceramide species, has been identified recently as the principal mediator of obesity-derived insulin resistance, impaired fatty acid oxidation, and hepatic steatosis. In this review, we have sought to cover the importance of the ceramide species and their metabolism, the main ceramide signaling pathways in obesity, and the link between C16:0 ceramide, FAO, and obesity.-Fucho, R., Casals, N., Serra, D., Herrero, L. Ceramides and mitochondrial fatty acid oxidation in obesity.

ABSTRACT T cells must tightly regulate their metabolic processes to cope with varying bioenergetic demands depending on their state of differentiation. The metabolic sensor AMPK is activated in states of low energy supply andmodulates cellular metabolism toward a catabolic state. Although this enzyme isknown tobeparticularly active in regulatory T (T reg ) cells, its impact on T helper (T h )‐cell differentiation is poorly understood. We investigated the impact of several AMPK activators on T reg ‐cell differentiation and found that the direct activator AICAR (5‐aminoimidazole‐4‐carboxamide ribonucleotide), but not the indirect activators metformin and 2‐deoxyglucose, strongly enhanced Treg‐cell induction by specifically enhancing T reg ‐cell expansion. Conversely, T h 17 generation was impaired by the agent. Further investigation of the metabolic background of our observations revealed that AICAR enhanced both cellular mitochondrogenesis and fatty acid uptake. Consistently, increased Treg induction was entirely reversible on inhibition of fatty acid oxidation, thus confirming the…

Cachexia is a devastating syndrome that causes morbidity and mortality in a large number of patients with cancer. However, the mechanisms of cancer cachexia remain poorly understood. Accumulation of misfolded proteins in the endoplasmic reticulum (ER) causes stress. The ER responds to this stress through activating certain pathways commonly known as the unfolding protein response (UPR). The main function of UPR is to restore homeostasis, but excessive or prolonged activation of UPR can lead to pathologic conditions. In this study, we examined the role of ER stress and UPR in regulation of skeletal muscle mass in naïve conditions and during cancer cachexia. Our results demonstrate that multiple markers of ER stress are highly activated in skeletal muscle of Lewis lung carcinoma (LLC) and Apc(Min/+) mouse models of cancer cachexia. Treatment of mice with 4-phenylbutyrate (4-PBA), a chemical chaperon and a potent inhibitor of ER stress, significantly reduced skeletal muscle strength and mass in both control and LLC-bearing mice. Blocking the UPR also increased the proportion of…

The functional integrity of the intestinal epithelial barrier relies on tight coordination of cell proliferation and migration, with failure to regulate these processes resulting in disease. It is not known whether cell proliferation is sufficient to drive epithelial cell migration during homoeostatic turnover of the epithelium. Nor is it known precisely how villus cell migration is affected when proliferation is perturbed. Some reports suggest that proliferation and migration may not be related while other studies support a direct relationship. We used established cell-tracking methods based on thymine analog cell labeling and developed tailored mathematical models to quantify cell proliferation and migration under normal conditions and when proliferation is reduced and when it is temporarily halted. We found that epithelial cell migration velocities along the villi are coupled to cell proliferation rates within the crypts in all conditions. Furthermore, halting and resuming proliferation results in the synchronized response of cell migration on the villi. We conclude that cell…

ABSTRACT Cardiac dysfunction develops during sepsis in humans and rodents. In the model of polymicrobial sepsis induced by cecal ligation and puncture (CLP), we investigated the role of the NLRP3 inflammasome in the heart. Mouse heart homogenates from sham‐procedure mice contained high mRNA levels of NLRP3 and IL‐1β. Usingthe inflamm a some protocol, exposure of cardiomyocytes (CMs) to LPS followed by ATP or nigericin caused release of mature IL‐1β. Immuno staining of left ventricular frozen sections before and 8 h after CLP revealed the presence of NLRP3 and IL‐1β proteins inCMs. CLP caused substantial increases in mRNAs for IL‐1β and NLRP3 in CMs which are reduced in the absence of either C5aR1 or C5aR2. After CLP, NLRP3 2/2 mice showed reduced plasma levels of IL‐1βand IL‐6. In vitro exposure of wild‐type CMs to recombinant C5a (rC5a) cause delevations in both cytosolic and nuclear/mitochondrial reactive oxygen species (ROS), which were C5a‐receptor dependent. Use of a selective NOX2 inhibitor prevented increased cytosolic and nuclear/mitochondrial ROS levels and release of…

ABSTRACT Articular cartilage has little regenerative capacity. Recently, genetic lineage tracing experiments have revealed chondrocyte progenitors at the articular surface. We further characterized these progenitors by using in vivo genetic approaches. Histone H2B–green fluorescent protein retention revealed that superficial cells divide more slowly than underlying articular chondrocytes. Clonal genetic tracing combined with immunohistochemistry revealed that superficial cells renew their number by symmetric division, express mesenchymal stem cell markers, and generate chondrocytes via both asymmetric and symmetric differentiation. Quantitative analysis of cellular kinetics, in combination with phosphotungstic acid–enhanced micro–computed tomography, showed that superficial cells generate chondrocytes and contribute to the growth and reshaping of articular cartilage. Furthermore, we found that cartilage renewal occurs as the progeny of superficial cells fully replace fetal chondrocytes during early postnatal life. Thus, superficial cells are self‐renewing progenitors that are…

The role of resolvins in abdominal aortic aneurysm (AAA) has not been established. We hypothesized that treatment with D-series resolvins (RvD2 or RvD1) would attenuate murine AAA formation through alterations in macrophage polarization and cytokine expression. Male C57/B6 mice (n = 9 per group) 8 to 12 wk old received RvD2 (100 ng/kg/treatment), RvD1 (100 ng/kg/treatment), or vehicle only every third day beginning 3 d before abdominal aortic perfusion with elastase as prevention. Aortas were collected 14 d after elastase perfusion. Cytokine analysis (n = 5 per group) or confocal microscopy (n = 4 per group) was performed. In a separate experiment, RvD2 was provided to mice with small AAAs 3 d after elastase treatment (n = 8 per group). Additionally, apolipoprotein E knockout mice treated with angiotensin II (1000 ng/kg) were treated with RvD2 or vehicle alone (n = 10 per group) in a nonsurgical model of AAA. To determine the effect of RvD2 on macrophage polarization, confocal staining for macrophages, M1 and M2 macrophage subtypes, α-actin, and DAPI was performed. Mean aortic…

Parkinson's disease (PD) is characterized by a progressive loss of dopaminergic neurons with limited treatment options. Emerging evidence shows that FTY720 protects against neural injury via modulation of the sphingosine-1-phosphate 1 receptor (S1PR1). However, it remains unclear whether FTY720 could influence neurodegeneration in PD. Therefore, the present study was designed to determine the impact of fingolimod (FTY720), a sphingosine-1-phosphate receptor (S1PR) agonist, on 2 mouse models of PD. We found that FTY720 significantly reduced the deficit of motor function, diminished the loss of tyrosine hydroxylase-positive neurons in the substantia nigra, and attenuated the decrease of striatal dopamine and metabolite levels in mice receiving 6-hydroxydopamine (6-OHDA) or rotenone to simulate PD. An S1PR1-selective antagonist, W146, blocked the neuroprotective effects of FTY720. Of note, FTY720 retained the phosphorylation of ERK, together with a decreased expression of cleaved caspase-3 in mice treated with 6-OHDA or rotenone. In vitro studies revealed that FTY720 also attenuated…

Apolipoprotein M (ApoM) transports sphingosine-1-phosphate (S1P) in plasma, and ApoM-deficient mice (Apom(-/-)) have ∼50% reduced plasma S1P levels. There are 5 known S1P receptors, and S1P induces adherens junction formation between endothelial cells through the S1P1 receptor, which in turn suppresses vascular leak. Increased vascular permeability is a hallmark of inflammation. The purpose of this study was to explore the relationships between vascular leakage in ApoM deficiency and S1P1 function in normal physiology and in inflammation. Vascular permeability in the lungs was assessed by accumulation of dextran molecules (70 kDa) and was increased ∼40% in Apom(-/-) mice compared to WT (C57Bl6/j) mice. Reconstitution of plasma ApoM/S1P or treatment with an S1P1 receptor agonist (SEW2871) rapidly reversed the vascular leakage to a level similar to that in WT mice, suggesting that it is caused by decreased plasma levels of S1P and reduced S1P1 stimulation. In a carrageenan-induced model of inflammation, Apom(-/-) mice had increased vascular leakage compared with that in WT mice.…

We examined the involvement of adipocyte cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2)-prostaglandin E receptor (EP)3-mediated signaling during hypertrophy and hypoxia in the development of obesity-associated adipose tissue (AT) inflammation and insulin resistance. The experiments were conducted with high-fat diet (HFD)-induced obese rats, db/db mice, human subjects, and 3T3-L1 and the human Simpson-Golabi-Behmel syndrome (SGBS) adipocytes; the groups were treated with selective inhibitors of COX-2 [celecoxib 30 mg/kg, half maximal inhibitory concentration (IC50) ≈ 0.04 µM] and EP3 (L-798106 100 µg/kg, IC50 ≈ 0.5 µM) or a short interfering RNA. There were strong, positive correlations between adipocyte COX-2 and EP3 gene expressions and the AT TNF-α and monocyte chemotactic protein-1 contents and the homeostatic model assessment for insulin resistance in HFD-induced obese rats, as well as body mass index in human subjects. Treatment with COX-2 and EP3 inhibitors significantly reversed AT inflammatory gene and protein expressions (-50%) and impaired glucose and insulin…

Age is, by far, the greatest risk factor for Alzheimer's disease (AD), yet few AD drug candidates have been generated that target pathways specifically associated with the aging process itself. Two ubiquitous features of the aging brain are the intracellular accumulation of aggregated proteins and inflammation. As intraneuronal amyloid protein is detected before markers of inflammation, we argue that old, age-associated, aggregated proteins in neurons can induce inflammation, resulting in multiple forms of brain toxicities. The consequence is the increased risk of old, age-associated, neurodegenerative diseases. As most of these diseases are associated with the accumulation of aggregated proteins, it is possible that any therapeutic that reduces intracellular protein aggregation will benefit all.-Currais, A., Fischer, W., Maher, P., Schubert, D. Intraneuronal protein aggregation as a trigger for inflammation and neurodegeneration in the aging brain.

) mice and rats with diet-induced hypercholesterolemia (DIHC) rats were used to explore the effects of hypercholesterolemia on the progression of OA. Both models exhibited OA-like changes, characterized primarily by a loss of proteoglycans, collagen and aggrecan degradation, osteophyte formation, changes to subchondral bone architecture, and cartilage degradation. Surgical destabilization of the knees resulted in a dramatic increase of degradative OA symptoms in animals fed a high-cholesterol diet compared with controls. Clinically relevant doses of free cholesterol resulted in mitochondrial dysfunction, overproduction of reactive oxygen species (ROS), and increased expression of degenerative and hypertrophic markers in chondrocytes and breakdown of the cartilage matrix. We showed that the severity of diet-induced OA changes could be attenuated by treatment with both atorvastatin and a mitochondrial targeting antioxidant. The protective effects of the mitochondrial targeting antioxidant were associated with suppression of oxidative damage to chondrocytes and restoration of…

The apolipoprotein E4 (E4) allele is present worldwide, despite its associations with higher risk of cardiovascular morbidity, accelerated cognitive decline during aging, and Alzheimer's disease (AD). The E4 allele is especially prevalent in some tropical regions with a high parasite burden. Equatorial populations also face a potential dual burden of high E4 prevalence combined with parasitic infections that can also reduce cognitive performance. We examined the interactions of E4, parasite burden, and cognitive performance in a traditional, nonindustrialized population of Amazonian forager‐horticulturalists ( N = 372) to test whether E4 protects against cognitive decline in environments with a heavy pathogen burden. Contrary to observations in industrial pop‐ulations, older adult E4 carriers with high parasite burdens either maintained or showed slight improvements in cognitive performance, whereas non‐E4 carriers with a high parasite burden showed reduced cognitive performance. Being an E4 carrier is the strongest risk factor to date of AD and cognitive decline in industrial…

There is strong diurnal variation in the symptoms and severity of chronic inflammatory diseases, such as rheumatoid arthritis. In addition, disruption of the circadian clock is an aggravating factor associated with a range of human inflammatory diseases. To investigate mechanistic links between the biological clock and pathways underlying inflammatory arthritis, mice were administered collagen (or saline as a control) to induce arthritis. The treatment provoked an inflammatory response within the limbs, which showed robust daily variation in paw swelling and inflammatory cytokine expression. Inflammatory markers were significantly repressed during the dark phase. Further work demonstrated an active molecular clock within the inflamed limbs and highlighted the resident inflammatory cells, fibroblast-like synoviocytes (FLSs), as a potential source of the rhythmic inflammatory signal. Exposure of mice to constant light disrupted the clock in peripheral tissues, causing loss of the nighttime repression of local inflammation. Finally, the results show that the core clock proteins…

ABSTRACT Selenocysteine is the 21st proteinogenic amino acid inmammals. The human genome contains 25 genes encoding selenoproteins, and their significance for human health is increasingly recognized through the identification of patients with inborn errors in selenoprotein biosynthetic factors or in individual selenoproteins. Mutations in selenoprotein N ( SEPN1 ) lead to a spectrum of disorders collectively called SEPN1 ‐related myopathy, and mutations in glutathione peroxidase 4 ( GPX4 ) cause respiratory failure and bone defects, and mutations in thioredoxin reductase 2 ( TXNRD2 ) are associated with familial glucocorticoid deficiency. Pathogenic mutations in selenocysteine synthase ( SEPSECS ) cause neurodevelopmental disorders, but also other factors epistatic to selenoprotein biosynthesis, such as SECIS‐binding protein 2 ( SECISBP2 ) and tRNA [Ser]Sec , are known to cause complex disorders. Mutations in the latter 2 genes involve impaired metabolism and action of thyroid hormones, which lead to delayed bone growth and maturation. Mutations in SECISBP2 sometimes affect nervous…

ABSTRACT Cannabinoids apparently act on inflammation through mechanisms different fromthose of agents such as nonsteroidal anti‐inflammatory drugs (NSAIDs). As a class, the cannabinoids are generally free fromthe adverse effects associated with NSAIDs. Their clinical development thus provides a new approach to treatment of diseases characterized by acute and chronic inflammation and fibrosis. A concise survey of the anti‐inflammatory actions of the phytocannabinoids ∆ 9 ‐tetrahydrocannabinol (THC), cannabidiol, cannabichromene, and cannabinol is presented. Mention is also made of the noncannabinoid plant components and pyrolysis products, followed by a discussion of 3 synthetic preparations—Cesamet (nabilone; Meda Pharmaceuticals, Somerset, NJ, USA), Marinol (dronabinol;THC; AbbVie, Inc., North Chicago, IL, USA), and Sativex ( Cannabis extract; GW Pharmaceuticals, Cambridge United Kingdom)—that have anti‐inflammatory effects. A fourth synthetic cannabinoid, ajulemic acid (AJA; CT‐3; Resunab; Corbus Pharmaceuticals, Norwood, MA, USA), is discussed in greater detail because it…

ABSTRACT Hypophosphatemia can lead to muscle weakness and respiratory and heart failure, but the mechanism is unknown. To address this question, we noninvasively assessed rates of muscle ATP synthesis in hypophosphatemic mice by using in vivo saturation transfer [ 31 P]‐magnetic resonance spectroscopy. By using this approach, we found that basal and insulin‐stimulated rates of muscle ATP synthetic flux (VATP) and plasma inorganic phosphate (Pi)were reduced by 50% in mice with diet‐induced hypophosphatemia as well as in sodium‐dependent P i transporter solute carrier family 34, member 1 ( NaPi2a )‐knockout ( NaPi2a ‐/‐ ) mice compared with their wild‐type littermate controls. Rates of V ATP normalized in both hypophosphatemic groups after restoring plasma P i concentrations. Furthermore, V ATP was directly related to cellular and mitochondrial P i uptake in L6 and RC13 rodent myocytes and isolated muscle mitochondria. Similar findings were observed in a patient with chronic hypophosphatemia as a result of a mutation in SLC34A3 who had a 50% reduction in both serum P i content and…

Podocytes play a key role in diabetic nephropathy pathogenesis, but alteration of their metabolism remains unknown in human kidney. By using a conditionally differentiating human podocyte cell line, we addressed the functional and molecular changes in podocyte energetics during in vitro development or under high glucose conditions. In 5 mM glucose medium, we observed a stepwise activation of oxidative metabolism during cell differentiation that was characterized by peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α)-dependent stimulation of mitochondrial biogenesis and function, with concomitant reduction of the glycolytic enzyme content. Conversely, when podocytes were cultured in high glucose (20 mM), stepwise oxidative phosphorylation biogenesis was aborted, and a glycolytic switch occurred, with consecutive lactic acidosis. Expression of the master regulators of oxidative metabolism transcription factor A mitochondrial, PGC-1α, AMPK, and serine-threonine liver kinase B1 was altered by high glucose, as well as their downstream signaling networks. Focused…

A disintegrin and metalloproteinase with thrombospondin type I motif (ADAMTS)2, 3, and 14 are collectively named procollagen N-proteinases (pNPs) because of their specific ability to cleave the aminopropeptide of fibrillar procollagens. Several reports also indicate that they could be involved in other biological processes, such as blood coagulation, development, and male fertility, but the potential substrates associated with these activities remain unknown. Using the recently described N-terminal amine isotopic labeling of substrate approach, we analyzed the secretomes of human fibroblasts and identified 8, 17, and 22 candidate substrates for ADAMTS2, 3, and 14, respectively. Among these newly identified substrates, many are components of the extracellular matrix and/or proteins related to cell signaling such as latent TGF-β binding protein 1, TGF-β RIII, and dickkopf-related protein 3. Candidate substrates for the 3 ADAMTS have been biochemically validated in different contexts, and the implication of ADAMTS2 in the control of TGF-β activity has been further demonstrated in human…

ABSTRACT Tendon injures are common orthopedic conditions, but tendon development and the pathogenesis of tendon injures, such as tendinopathy, remain largely unknown and have limited the development of clinical therapy. Studies on tenogenic differentiation at the molecular level may help in developing novel therapeutic strategies. As novel regulators, long noncoding RNAs (lncRNAs) have been found to have widespread biological functions, and emerging evidence demonstrates that lncRNAs may play important regulatory roles in cell differentiation and tissue regeneration. In this study, we found that lncRNA H19 stimulated tenogenesis of human tendon‐derived stem cells. Stable overexpression of H19 significantly accelerated TGF‐b1‐induced tenogenic differentiation in vitro and accelerated tendon healing in a mouse tendon defect model. H19 directly targeted miR‐29b‐3p, which is considered to be a negative regulator of tenogenesis. Furthermore, miR‐29b‐3p directly suppressed the expression of TGF‐β1 and type I collagen, thereby forming a novel regulatory feedback loop between H19 and TGF‐b1…

Nicotinic α‐7 acetylcholine receptor (nAChRα7) is a critical regulator of cholinergic anti‐inflammatory actions in several diseases, including acute respiratory distress syndrome(ARDS). Given the potential importance of α7nAChR as a therapeutic target, we evaluated whether PNU‐282987, an α7nAChR agonist, is effective in protecting the lung against inflammation. We performed intratracheal instillation of LPS to generate acute lung injury (ALI) in C57BL/6 mice. PNU‐282987 treatment, either before or after ALI induction, reduced neutrophil recruitment and IL‐ 1β, TNF‐α, IL‐6, keratinocyte chemoattractant (KC), and IL‐10 cytokine levels in the bronchoalveolar lavage fluid ( P > 0.05). In addition, lung NF‐κB phosphorylation decreased, along with collagen fiber deposition and the number of matrix metalloproteinase‐9 + and −2 + cells, whereas the number of tissue inhibitor of metalloproteinase‐1+ cells increased ( P < 0.05). PNU‐282987 treatment also reduced lung mRNA levels and the frequency of M1 macrophages, whereas cells expressing the M2‐related markers CD206 and IL‐10 increased,…

In the debates surrounding biotechnology and genetically modified (GM) food, data from consumer polls are often presented as evidence for precaution and labeling. But how much do consumers actually know about the issue? New data collected from a nationwide U.S. survey reveal low levels of knowledge and numerous misperceptions about GM food. Nearly equal numbers of consumers prefer mandatory labeling of foods containing DNA as do those preferring mandatory labeling of GM foods. When given the option, the majority of consumers prefer that decisions about GM food be taken out of their hands and be made by experts. After answering a list of questions testing objective knowledge of GM food, subjective, self-reported knowledge declines somewhat, and beliefs about GM food safety increase slightly. Results suggest that consumers think they know more than they actually do about GM food, and queries about GM facts cause respondents to reassess how much they know. The findings question the usefulness of results from opinion polls as a motivation for creating public policy surrounding GM…

Obesity is associated with chronic low-grade inflammation, characterized by leukocytosis and inflammation in the adipose tissue. Continuous activation of the immune system is a stressor for hematopoietic stem and progenitor cells (HSPCs) in the bone marrow (BM). Here we studied how diet-induced obesity (DIO) affects HSPC population dynamics in the BM. Eight groups of age-matched C57Bl/6 mice received a high-fat diet (45% kilocalories from fat) ranging from 1 d up to 18 wk. The obesogenic diet caused decreased proliferation of lineage(-)Sca-1(+)c-Kit(+) (LSK) cells in the BM and a general suppression of progenitor cell populations including common lymphoid progenitors and common myeloid progenitors. Within the LSK population, DIO induced a shift in stem cells that are capable of self-renewal toward maturing multipotent progenitor cells. The higher differentiation potential resulted in increased lymphoid and myeloid ex vivo colony-forming capacity. In a competitive BM transplantation, BM from obese animals showed impaired multilineage reconstitution when transplanted into chow-fed…

The mechanism of blood-brain barrier (BBB) disruption, involved in poststroke edema and hemorrhagic transformation, is important but elusive. We investigated microRNA-150 (miR-150)-mediated mechanism in the disruption of BBB after stroke in rats. We found that up-regulation of miR-150 increased permeability of BBB as detected by MRI after permanent middle cerebral artery occlusion in vivo as well as increased permeability of brain microvascular endothelial cells after oxygen-glucose deprivation in vitro. The expression of claudin-5, a key tight junction protein, was decreased in the ischemic boundary zone after up-regulation of miR-150. We found in brain microvascular endothelial cells that overexpression of miR-150 decreased not only cell survival rate but also the expression levels of claudin-5 after oxygen-glucose deprivation. With dual-luciferase assay, we confirmed that miR-150 could directly regulate the angiopoietin receptor Tie-2. Moreover, silencing Tie-2 with lentivirus-delivered small interfering RNA reversed the effect of miR-150 on endothelial permeability, cell…

Platelet-derived exosomes mediate platelet atherogenic interactions with endothelial cells and monocytes. A new method for isolation of plasma platelet-derived exosomes is described and used to examine effects of aging and aspirin on exosome cargo proteins. Exosome secretion by purified platelets in vitro did not increase after exposure to thrombin or collagen, as assessed by exosome counts and quantification of the CD81 exosome marker. Thrombin and collagen increased exosome content of α-granule chemokines CXCL4 and CXCL7 and cytoplasmic high-mobility group box 1 (HMGB1) protein, but not membrane platelet glycoprotein VI (GPVI), with dependence on extracellular calcium. Aspirin consumption significantly blocked thrombin- and collagen-induced increases in exosome cargo levels of chemokines and HMGB1, without altering total exosome secretion or GPVI cargo. Plasma platelet-derived exosomes, enriched by absorption with mouse antihuman CD42b [platelet glycoprotein Ib (GPIb)] mAb, had sizes and cargo protein contents similar to those of exosomes from purified platelets. The plasma…

ABSTRACT In contrast to the well‐characterized effects of specialized proresolving lipid mediators (SPMs) derived fromeicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), little is known about themetabolic fate of the intermediary long‐chain (LC) n‐3 polyunsaturated fatty acid (PUFA) docosapentaenoic acid (DPA). In this double blind crossover study, shifts in circulating levels of n‐3 and n‐6 PUFA‐derived bioactive lipid mediators were quantified by an unbiased liquid chromatography‐tandem mass spectrometry lipidomic approach. Plasma was obtainedfrom humansubjects before andafter 7 d of supplementationwith pure n‐3DPA, n‐3 EPA or placebo (olive oil). DPA supplementation increased the SPM resolvin D5 n‐3DPA (RvD5 n‐3DPA ) and maresin (MaR)‐1, the DHA vicinal diol 19, 20‐dihydroxy‐DPA and n‐6 PUFA derived 15‐keto‐PG E 2 (15‐keto‐PGE 2 ). EPA supplementation had no effect on any plasma DPA or DHA derived mediators, but markedly elevated monohydroxy‐eicosapentaenoic acids (HEPEs), including the e ‐series resolvin (RvE) precursor 18‐HEPE; effects not observed with DPA…

Exercise promotes multiple beneficial effects on muscle function, including induction of mitochondrial biogenesis. miR-133a is a muscle-enriched microRNA that regulates muscle development and function. The role of miR-133a in exercise tolerance has not been fully elucidated. In the current study, mice that were deficient in miR-133a demonstrated low maximal exercise capacity and low resting metabolic rate. Transcription of the mitochondrial biogenesis regulators peroxisome proliferator-activated receptor-γ coactivator 1-α, peroxisome proliferator-activated receptor-γ coactivator 1-β, nuclear respiratory factor-1, and transcription factor A, mitochondrial were lower in miR-133a-deficient muscle, which was consistent with lower mitochondrial mass and impaired exercise capacity. Six weeks of endurance exercise training increased the transcriptional level of miR-133a and stimulated mitochondrial biogenesis in wild-type mice, but failed to improve mitochondrial function in miR-133a-deficient mice. Further mechanistic analysis showed an increase in the miR-133a potential target, IGF-1…

ABSTRACT In this study, we strove to substantiate the ability of linc‐MAF‐4 to act as a regulator of pathogenesis during multiple sclerosis (MS). We recruited 34 patients who were diagnosed with MS according to the revised McDonald criteria. Six patients with MS and 5 healthy volunteers contributed peripheral blood mononuclear cells for microarray analysis. Subsequent knockdown and overexpression of linc‐MAF‐4 in naive CD4 + T cells from the additional 28 patients with MS was performed to track changes in CD4 + T‐cell subsets and their function, as well as to confirm results from the prior microarray analysis. Expression of linc‐MAF‐4 increased significantly in peripheral blood mononuclear cells of patients with MS compared with those of control participants. In addition, linc‐MAF‐4 regulated encephalitogenic T helper (T h )1‐cell differentiation in patients with MS. Transfection of synthetic linc‐MAF‐4 into naive CD4 + T cells facilitated T h 1‐cell differentiation and inhibited T h 2‐cell differentiation by directly inhibiting MAF, which is a T h 2‐cell transcription factor.…

Insulin secretion by pancreatic β-cells is primarily regulated by glucose; however, hormones and additional nutrients, such as long-chain fatty acids, also play an important role in adjusting insulin output to physiologic needs. To examine the role of short-chain fatty acids (SCFAs) in β-cell function, we analyzed mouse models of gain and loss of function of GPR41 (FFAR3), a receptor for SCFAs, vs. wild-type control mice. GPR41 gain of function [GPR41-overexpressing transgenic (41 Tg) model] and GPR41 loss of function [GPR41-knockout (KO 41) model] resulted in complementary changes in glucose tolerance, without significant effects on insulin sensitivity. KO 41 mice showed fasting hypoglycemia, which was consistent with increased basal and glucose-induced insulin secretion by islets in vitro Mirroring this, 41 Tg islets showed impaired glucose responsiveness in vitro Microarray analysis of islets from 41 Tg mice indicated significant alterations in gene expression patterns; several of the altered genes were chosen for further analysis and were also observed to change upon incubation…

Complement activation, an integral arm of innate immunity, may be the critical link to the pathogenesis of idiopathic pulmonary fibrosis (IPF). Whereas we have previously reported elevated anaphylatoxins-complement component 3a (C3a) and complement component 5a (C5a)-in IPF, which interact with TGF-β and augment epithelial injury in vitro, their role in IPF pathogenesis remains unclear. The objective of the current study is to determine the mechanistic role of the binding of C3a/C5a to their respective receptors (C3aR and C5aR) in the progression of lung fibrosis. In normal primary human fetal lung fibroblasts, C3a and C5a induces mesenchymal activation, matrix synthesis, and the expression of their respective receptors. We investigated the role of C3aR and C5aR in lung fibrosis by using bleomycin-injured mice with fibrotic lungs, elevated local C3a and C5a, and overexpression of their receptors via pharmacologic and RNA interference interventions. Histopathologic examination revealed an arrest in disease progression and attenuated lung collagen deposition (Masson's trichrome,…

ABSTRACT BMAL1, the nonredundant transcription factor in the core molecular clock, has been implicated in cardiometabolic diseases in mice and humans. BMAL1 controls the cyclic trafficking of Ly6c hi monocytes to sites of acute inflammation. Myeloid deficiency of Bmal1 also worsens chronic inflammation in diet‐induced obesity. We studied whether myeloid Bmal1 deletion promotes atherosclerosis by enhancing monocyte recruitment to atherosclerotic lesions. By generating Bmal1 FloxP/FloxP ;LysM Cre mice on the Apoe −/− background, we showed that Bmal1 deletion in myeloid cells increased the size of atherosclerotic lesions. Bmal1 deficiency in monocytes and macrophages resulted in an increased total number of lesional macrophages in general and Ly6c hi infiltrating monocyte‐macrophages in particular, accompanied by skewed M2 to M1 macrophage phenotype. Ly6c hi and/or Ly6c l ° monocyte subsets in blood, spleen, and bone marrow were not altered. Cell tracking and adoptive transfer of Ly6c hi monocytes showed Bmal1 deficiency induced more trafficking of Ly6c hi monocytes to atherosclerotic…

ABSTRACT Potent and selective chemical probes are valuable tools for discovery of novel treatments for human diseases. NF‐κβ‐inducing kinase (NIK) is a key trigger in the development of liver injury and fibrosis. Whether inhibition of NIK activity by chemical probes ameliorates liver inflammation and injury is largely unknown. In this study, a small‐molecule inhibitor of NIK, B022, was found to be a potent and selective chemical probe for liver inflammation and injury. B022 inhibited the NIK signaling pathway, including NIK‐induced p100‐to‐p52 processing and inflammatory gene expression, both in vitro and in vivo. Furthermore, in vivo administration of B022 protected against not only NIK but also CCl 4 ‐induced liver inflammation and injury. Our data suggest that inhibition of NIK is a novel strategy for treatment of liver inflammation, oxidative stress, and injury.—Ren, X., Li, X., Jia, L., Chen, D., Hou, H., Rui, L., Zhao, Y., Chen, Z. A small‐molecule inhibitor of NF‐κβ‐inducing kinase (NIK) protects liver from toxin‐induced inflammation, oxidative stress, and injury. FASEB J.…

ABSTRACT Cannabinoids modulate intestinal permeability through cannabinoid receptor 1 (CB1). The endocannabinoid‐like compounds oleoylethanolamine (OEA) and palmitoylethanolamine (PEA) play an important role in digestive regulation, and we hypothesized they would also modulate intestinal permeability. Transepithelial electrical resistance (TEER) was measured in human Caco‐2 cells to assess permeability after application of OEA and PEA and relevant antagonists. Cells treated with OEA and PEA were stained for cytoskeletal F‐actin changes and lysed for immunoassay. OEA and PEA were measured by liquid chromatography‐tandem mass spectrometry. OEA (applied apically, logEC50 25.4) and PEA (basolaterally, logEC50 24.9; apically logEC50 25.3) increased Caco‐2 resistance by 20–30% via transient receptor potential vanilloid (TRPV)‐1 and peroxisome proliferator‐activated receptor (PPAR)‐a. Preventing their degradation (by inhibiting fatty acid amide hydrolase) enhanced the effects of OEA and PEA. OEA and PEA induced cytoskeletal changes and activated focal adhesion kinase and ERKs 1/2, and…

We aimed to characterize antimicrobial zinc trafficking within macrophages and to determine whether the professional intramacrophage pathogen Salmonella enterica serovar Typhimurium (S Typhimurium) subverts this pathway. Using both Escherichia coli and S Typhimurium, we show that TLR signaling promotes the accumulation of vesicular zinc within primary human macrophages. Vesicular zinc is delivered to E. coli to promote microbial clearance, whereas S. Typhimurium evades this response via Salmonella pathogenicity island (SPI)-1. Even in the absence of SPI-1 and the zinc exporter ZntA, S Typhimurium resists the innate immune zinc stress response, implying the existence of additional host subversion mechanisms. We also demonstrate the combinatorial antimicrobial effects of zinc and copper, a pathway that S. Typhimurium again evades. Our use of complementary tools and approaches, including confocal microscopy, direct assessment of intramacrophage bacterial zinc stress responses, specific E. coli and S Typhimurium mutants, and inductively coupled plasma mass spectroscopy, has enabled…

Matricellular proteins mediate pleiotropic effects during tissue injury and repair. CCN1 is a matricellular protein that has been implicated in angiogenesis, inflammation, and wound repair. In this study, we identified CCN1 as a gene that is differentially up-regulated in alveolar mesenchymal cells of human subjects with rapidly progressive idiopathic pulmonary fibrosis (IPF). Elevated levels of CCN1 mRNA were confirmed in lung tissues of IPF subjects undergoing lung transplantation, and CCN1 protein was predominantly localized to fibroblastic foci. CCN1 expression in ex vivo IPF lung fibroblasts correlated with gene expression of the extracellular matrix proteins, collagen (Col)1a1, Col1a2, and fibronectin as well as the myofibroblast marker, α-smooth muscle actin. RNA interference (RNAi)-mediated knockdown of CCN1 down-regulated the constitutive expression of these profibrotic genes in IPF fibroblasts. TGF-β1, a known mediator of tissue fibrogenesis, induces gene and protein expression of CCN1 via a mothers against decapentaplegic homolog 3 (SMAD3)-dependent mechanism.…

ABSTRACT Macrophage activation is an important feature of primary biliary cholangitis (PBC) pathogenesis and other cholestatic liver diseases. Galectin‐3 (Gal3), a pleiotropic lectin, is produced by monocytic cells and macrophages. However, its role in PBC has not been addressed. We hypothesized that Gal3 is a key to induce NOD‐like receptor family, pyrin domain containing 3 (NLRP3) inflammasome in macrophages and in turn to propagate proinflammatory IL‐17 signaling. In liver tissues from patients with PBC and dnTGF‐βRII mice, a model of autoimmune cholangitis, the expression of Gal3, NLRP3, and the adaptor protein adaptor apoptosis‐associated speck like protein was induced, with the downs tream activation of caspase‐1 and IL‐1β. Inwild‐typehepaticmacrophages, deoxycholic acid induced the association of Gal3 and NLRP3with direct activation of the inflammasome, resulting in an increase in IL‐1β. Downstreamretinoid‐related orphan receptor CmRNA, IL‐17A, and IL‐17Fwere induced. In Gal3 ‐/‐ macrophages, no inflammasome activation was detected. To confirm the key role of Gal3 in the…

ABSTRACT Reduced oxygen (O 2 ) levels (hypoxia) are present during embryogenesis and exposure to altitude and in pathologic conditions. During embryogenesis, myogenic progenitor cells reside in a hypoxic micro environment, which may regulate their activity. Satellite cells are myogenic progenitor cells localized in a local environment, suggesting that the O 2 level could affect their activity during muscle regeneration. In this review, we present the idea that O 2 levels regulate myogenesis and muscle regeneration, we elucidate the molecular mechanisms under lying myogenesis and muscle regeneration in hypoxia and depict the rapeutic strategies using changes in O 2 levels to promote muscle regeneration. Severe hypoxia (£1% O 2 ) appears detrimental for myogenic differentiation in vitro, whereas a 3–6%O 2 level could promotemyogenesis. Hypoxia impairs the regenerative capacity of injured muscles. Although it remains to be explored, hypoxia may contribute to the muscle damage observed in patients with pathologies associated with hypoxia (chronic obstructive pulmonary disease, and…

)-dependent enzymes that catalyze post-translational modifications of proteins. Together, they regulate crucial cellular functions and are traditionally associated with aging and longevity. Dysregulation of sirtuins plays an important role in major diseases, including cancer and metabolic, cardiac, and neurodegerative diseases. They are extensively regulated in response to a wide range of stimuli, including nutritional and metabolic challenges, inflammatory signals or hypoxic and oxidative stress. Each sirtuin is regulated individually in a tissue- and cell-specific manner. The control of sirtuin expression involves all the major points of regulation, including transcriptional and post-translational mechanisms and microRNAs. Collectively, these mechanisms control the protein levels, localization, and enzymatic activity of sirtuins. In many cases, the regulators of sirtuin expression are also their substrates, which lead to formation of intricate regulatory networks and extensive feedback loops. In this review, we highlight the mechanisms mediating the physiologic and pathologic…

Small leucine-rich proteoglycans are emerging as important regulatory proteins within the extracellular matrix, where they exert both structural and nonstructural functions and hence are modulators of numerous biological processes, such as inflammation, fibrosis, and cell proliferation. One proteoglycan in particular, osteoglycin (OGN), also known as mimecan, shows great structural and functional diversity in normal physiology and in disease states, therefore making it a very interesting candidate for the development of novel therapeutic strategies. Unfortunately, the literature on OGN is confusing, as it has different names, and different transcript and protein variants have been identified. This review will give a clear overview of the different structures and functions of OGN that have been identified to date, portray its central role in pathophysiology, and highlight the importance of posttranslational processing, such as glycosylation, for the diversity of its functions.-Deckx, S., Heymans, S., Papageorgiou, A.-P. The diverse functions of osteoglycin: a deceitful dwarf, or a…

Differentiation of muscle satellite cells (MSCs) involves interaction of the proteins present in the extracellular matrix (ECM) with MSCs to regulate their activity, and therefore phenotype. Herein, we report fibromodulin (FMOD), a member of the proteoglycan family participating in the assembly of ECM, as a novel regulator of myostatin (MSTN) during myoblast differentiation. In addition to having a pronounced effect on the expression of myogenic marker genes [myogenin (MYOG) and myosin light chain 2 (MYL2)], FMOD was found to maintain the transcriptional activity of MSTN Moreover, coimmunoprecipitation and in silico studies performed to investigate the interaction of FMOD helped confirm that it antagonizes MSTN function by distorting its folding and preventing its binding to activin receptor type IIB. Furthermore, in vivo studies revealed that FMOD plays an active role in healing by increasing satellite cell recruitment to sites of injury. Together, these findings disclose a hitherto unrecognized regulatory role for FMOD in MSCs and highlight new mechanisms whereby FMOD circumvents…

Skeletal muscle possesses the ability to regenerate after injury, but this ability is impaired or delayed with aging. Regardless of age, muscle retains the ability to positively respond to stimuli, such as exercise. We examined whether exercise is able to improve regenerative response in skeletal muscle of aged mice. Twenty-two-month-old male C57Bl/6J mice (n = 20) underwent an 8-wk progressive exercise training protocol [old exercised (O-Ex) group]. An old sedentary (O-Sed) and a sedentary young control (Y-Ctl) group were included. Animals were subjected to injections of cardiotoxin into the tibialis anterior muscle. The tibialis anterior were harvested before [O-Ex/O-Sed/Y-Ctl control (CTL); n = 6], 10 d (O-Ex/O-Sed/Y-Ctl d 10; n = 8), and 28 d (O-Ex/O-Sed/Y-Ctl d 28; n = 6) postinjection. Average fiber cross-sectional area was reduced in all groups at d 10 (CTL: O-Ex: 2499 ± 140; O-Sed: 2320 ± 165; Y-Ctl: 2474 ± 269; d 10: O-Ex: 1191 ± 100; O-Sed: 1125 ± 99; Y-Ctl: 1481 ± 167 µm(2); P < 0.05), but was restored to control values in O-Ex and Y-Ctl groups at d 28 (O-Ex: 2257 ± 181;…

Mesenchymal stromal cell (MSC) therapy demands the attention of clinicians and scientists because of its potential in clinical fields that are bereft of medical options, but also because of the controversies that underlie its mode of action. MSCs are potent immune modulators, yet their biologic activity may not be innate, requiring licensing by their microenvironment. This property has prompted researchers to explore unique ways in which MSCs may be able to exert distinct biologic effects in different pathologic settings. More than 400 clinical trials have investigated the therapeutic capacity of MSCs in different pathologies, including liver disease. Along with their anti-inflammatory action, there are data to suggest that MSCs may exert direct antifibrotic effects, although enthusiasm for their use in patients has been tempered by concerns of a possible profibrotic role of endogenous MSCs in response to injury. There is a significant need for antifibrotic therapy to combat the increasing burden of patients with cirrhosis, and a concerted effort is required to determine the…

ABSTRACT Long intergenic noncoding RNAs (lincRNAs) are long noncoding transcripts (>200 nt) from the intergenic regions of annotated protein‐coding genes. We report here that the lincRNA gene lincRNA‐Tnfaip3 , located at mouse chromosome 10 proximal to the tumor necrosis factor α‐induced protein 3 ( Tnfaip3 ) gene, is an early‐primary response gene controlled by nuclear factor‐κB (NF‐κB) signaling in murine macrophages. Functionally, lincRNA‐ Tnfaip3 appears to mediate both the activation and repression of distinct classes of inflammatory genes in macrophages. Specifically, induction of lincRNA‐Tnfaip3 is required for the transactivation of NF‐κB‐regulated inflammatory genes in response to bacterial LPSs stimulation. LincRNA‐Tnfaip3 physically interacts with the high‐mobility group box 1 (Hmgb1), assembling a NF‐κB/Hmgb1/lincRNA‐Tnfaip3 complex in macrophages after LPS stimulation. This resultant NF‐κB/Hmgb1/lincRNA‐Tnfaip3 complex can modulate Hmgb1‐associated histone modifications and, ultimately, transactivation of inflammatory genes in mouse macrophages in response to microbial…

In humans, insulin sensitivity varies according to time of day, with decreased values in the evening and at night. Mechanisms responsible for the diurnal variation in insulin sensitivity are unclear. We investigated whether human adipose tissue (AT) expresses intrinsic circadian rhythms in insulin sensitivity that could contribute to this phenomenon. Subcutaneous and visceral AT biopsies were obtained from extremely obese participants (body mass index, 41.8 ± 6.3 kg/m(2); 46 ± 11 y) during gastric-bypass surgery. To assess the rhythm in insulin signaling, AKT phosphorylation was determined every 4 h over 24 h in vitro in response to different insulin concentrations (0, 1, 10, and 100 nM). Data revealed that subcutaneous AT exhibited robust circadian rhythms in insulin signaling (P < 0.00001). Insulin sensitivity reached its maximum (acrophase) around noon, being 54% higher than during midnight (P = 0.009). The amplitude of the rhythm was positively correlated with in vivo sleep duration (r = 0.53; P = 0.023) and negatively correlated with in vivo bedtime (r = -0.54; P = 0.020). No…

Receptor mediated transcytosis harnessing the cellular uptake and transport of natural ligands across the blood-brain barrier (BBB) has been identified as a means for antibody delivery to the CNS. In this study, we characterized bispecific antibodies in which a BBB-crossing antibody fragment FC5 was used as a BBB carrier. Cargo antibodies were either a high-affinity, selective antibody antagonist of the metabotropic glutamate receptor-1 (BBB-mGluR1), a widely abundant CNS target, or an IgG that does not bind the CNS target (BBB-NiP). Both BBB-NiP and BBB-mGluR1 demonstrated a similar 20-fold enhanced rate of transcytosis across an in vitro BBB model compared with mGluR1 IgG fused to a control antibody fragment. All 3 bispecific antibodies exhibited identical pharmacokinetics in vivo Comparative assessment of BBB-NiP and BBB-mGluR1 revealed that, whereas their serum pharmacokinetics and BBB penetration were identical, their central disposition (brain levels) and elimination (cerebrospinal fluid levels) were widely different, due to central target-mediated removal of the…

Monocyte/macrophages of patients with mild cognitive impairment (MCI) and Alzheimer disease (AD) are defective in phagocytosis and degradation amyloid β 1–42 (Aβ 1–42 ), but are improved by ω‐3 fatty acids (ω‐3s). The hypothesis of this study was that active Aβ 1–42 phagocytosis by macrophages prevents brain amyloidosis and thus maintains cognition. We studied the effects of self‐supplementation with a drink with v‐3s, antioxidants, and resveratrol on Mini‐Mental State Examination (MMSE) scores, macrophage M1M2 phenotype [the ratio of inflammatory cluster of differentiation (CD)54+CD80 and proresolution markers CD163+CD206], and Aβ 1–42 phagocytosis in patients initially diagnosed as having MCI or subjective cognitive impairment (SCI). At baseline, themedian MMSE score in patients in both the apolipoprotein E (ApoE) ε3/ε3 and ApoE ε3/ε4 groups was 26.0 and macrophage Aβ 1–42 phagocytos is was defective. The MMS Erate of change increased in the ApoE ε3/ε3 group a median 2.2 points per year ( P =0.015 compared to 0) but did not change in the ApoE ε3/ε4 group(P = 0.014 between groups).…

TGF-β is important in lung injury and remodeling processes. TGF-β and Wingless/integrase-1 (WNT) signaling are interconnected; however, the WNT ligand-receptor complexes involved are unknown. Thus, we aimed to identify Frizzled (FZD) receptors that mediate TGF-β-induced profibrotic signaling. MRC-5 and primary human lung fibroblasts were stimulated with TGF-β1, WNT-5A, or WNT-5B in the presence and absence of specific pathway inhibitors. Specific small interfering RNA was used to knock down FZD8. In vivo studies using bleomycin-induced lung fibrosis were performed in wild-type and FZD8-deficient mice. TGF-β1 induced FZD8 specifically via Smad3-dependent signaling in MRC-5 and primary human lung fibroblasts. It is noteworthy that FZD8 knockdown reduced TGF-β1-induced collagen Iα1, fibronectin, versican, α-smooth muscle (sm)-actin, and connective tissue growth factor. Moreover, bleomycin-induced lung fibrosis was attenuated in FZD8-deficient mice in vivo Although inhibition of canonical WNT signaling did not affect TGF-β1-induced gene expression in vitro, noncanonical WNT-5B mimicked…

Foot-and-mouth disease is a frequently occurring disease of cloven-hoofed animals that is caused by infection with the foot-and-mouth virus (FMDV). FMDV circumvents the type-I IFN response by expressing proteins that antagonize cellular innate immunity, such as leader protease and 3C protease. We identified the FMDV structural protein VP3 as a negative regulator of the virus-triggered IFN-β signaling pathway. Expression of FMDV VP3 inhibited the Sendai virus-triggered activation of IFN regulatory factor-3 and the expression of retinoic acid-inducible gene-I/melanoma differentiation-associated protein-5. Transient transfection and coimmunoprecipitation confirmed that the structural protein VP3 interacts with virus-induced signaling adapter (VISA), which is dependent on the C-terminal aa 111-220 of VP3. In addition, we found that FMDV VP3 inhibits the expression of VISA by disrupting its mRNA. Taken together, our findings reveal a novel strategy used by the structural VP3 protein of FMDV to evade host innate immunity.-Li, D., Yang, W., Yang, F., Liu, H., Zhu, Z., Lian, K., Lei, C.,…

ABSTRACT The cytokine IL‐10 has potent antifibrotic effects in models of adult fibrosis, but the mechanisms of action are unclear. Here, we report a novel finding that IL‐10 triggers a signal transducer and activator of transcription 3(STAT3)–dependent signaling pathway that regulates hyaluronan (HA) metabolism and drives adult fibroblasts to synthesize an HA‐rich pericellular matrix, which mimics the fetal regenerative wound healing phenotype with reduced fibrosis. By using cre‐lox‐mediated novel, inducible, fibroblast‐, keratinocyte‐, and wound‐specific STAT3‐knockdown postnatal mice—plus syngeneic fibroblast cell‐transplant models—we demonstrate that the regenerative effects of IL‐10 in postnatal wounds are dependent on HA synthesis and fibroblast‐specific STAT3‐dependent signaling. The importance of IL‐10‐induced HA synthesis for regenerative wound healing is demonstrated by inhibition of HA synthesis in a murine wound model by administering 4‐methylumbelliferone. Although IL‐10 and STAT3 signaling were intact, the antifibrotic repair phenotype that is induced by IL‐10…

ABSTRACT Autism spectrum disorder (ASD) is associated with physiological abnormalities, including abnormal redox and mitochondrial metabolism. Lymphoblastoid cell lines (LCLs) from some children with ASD exhibit increased oxidative stress, decreased glutathione redox capacity, and highly active mitochondria with increased vulnerability to reactive oxygen species (ROS). Because unaffected siblings (Sibs) of individuals with ASD share some redox abnormalities, we sought to determine whether LCLs from Sibs share ASD‐associated mitochondrial abnormalities. We evaluated mitochondrial bioenergetics in 10 sets of LCLs from children with ASD, Sibs, and unrelated/unaffected controls (Cons) after acute increases in ROS. Additionally, intracellular glutathione and uncoupling protein 2 ( UCP 2) gene expressions were quantified. Compared to Sib LCLs, ASD LCLs exhibited significantly higher ATP‐linked respiration, higher maximal and reserve respiratory capacity, and greater glycolysis and glycolytic reserve. ASD LCLs exhibited a significantly greater change in these parameters, with acute…

Talin is a ubiquitous, large focal adhesion protein that links intracellular networks with the extracellular matrix (ECM) via its connection with the actin cytoskeleton and membrane integrins. It is one of a handful molecules that can expose new recognition sites when undergoing force-induced mechanical unfolding, and it can bind and recruit cytoskeletal proteins that are involved in mechanotransduction. Talin has attracted great interest in the field of mechanobiology because of its plasticity in undergoing conformational changes under force stimulation as well as its cellular localization that bridges the cytoskeleton with the ECM. In addition to these roles in healthy cells, the dysregulation of talin activators can lead to disease states in which aberrant integrin activation and mechanotransduction precipitate changes in cell spreading, migration, and survival. New data have implicated a role for talin in diseases that are highly regulated by mechanical cues. In this review, we present the current understanding of talin structure, its relationship to binding partners, and its…

Alterations in sphingolipid metabolism, especially ceramide and sphingosine 1-phosphate, have been linked to colon cancer, suggesting that enzymes of sphingolipid metabolism may emerge as novel regulators and targets in colon cancer. Neutral ceramidase (nCDase), a key enzyme in sphingolipid metabolism that hydrolyzes ceramide into sphingosine, is highly expressed in the intestine; however, its role in colon cancer has not been defined. Here we show that molecular and pharmacological inhibition of nCDase in colon cancer cells increases ceramide, and this is accompanied by decreased cell survival and increased apoptosis and autophagy, with minimal effects on noncancerous cells. Inhibition of nCDase resulted in loss of β-catenin and inhibition of ERK, components of pathways relevant for colon cancer development. Furthermore, inhibition of nCDase in a xenograft model delayed tumor growth and increased ceramide while decreasing proliferation. It is noteworthy that mice lacking nCDase treated with azoxymethane were protected from tumor formation. Taken together, these studies show that…

Peroxiredoxin 6 (Prdx6) is essential for activation of NADPH oxidase type 2 (NOX2) in pulmonary microvascular endothelial cells (PMVECs), alveolar macrophages (AMs), and polymorphonuclear leukocytes. Angiotensin II and phorbol ester increased superoxide/H2O2 generation in PMVECs, AMs, and isolated lungs from wild-type (WT) mice, but had much less effect on cells or lungs from Prdx6-null or Prdx6-D140A-knock-in mice that lack the phospholipase A2 activity (PLA2) of Prdx6; addition of either lysophosphatidylcholine (LPC) or lysophosphatidic acid (LPA) to cells restored their oxidant generation. The generation of LPC by PMVECs required Prdx6-PLA2 We propose that Prdx6-PLA2 modulates NOX2 activation by generation of LPC that is converted to LPA by the lysophospholipase D activity of autotaxin (ATX/lysoPLD). Inhibition of lysoPLD with HA130 (cells,10 μM; lungs, 20 μM; IC50, 29 nM) decreased agonist-induced oxidant generation. LPA stimulates pathways regulated by small GTPases through binding to G-protein-coupled LPA receptors (LPARs). The LPAR blocker Ki16425 (cells, 10 μM; lungs, 25 μM;…

TGF-β plays a central role in the pathogenesis of fibroproliferative disorders. Defining the exact underlying molecular basis is therefore critical for the development of viable therapeutic strategies. Here, we show that expression of the facilitative glucose transporter 1 (GLUT1) is induced by TGF-β in fibroblast lines and primary cells and is required for the profibrotic effects of TGF-β. In addition, enhanced GLUT1 expression is observed in fibrotic areas of lungs of both patients with idiopathic pulmonary fibrosis and mice that are subjected to a fibrosis-inducing bleomycin treatment. By using pharmacologic and genetic approaches, we demonstrate that up-regulation of GLUT1 occurs via the canonical Smad2/3 pathway and requires autocrine activation of the receptor tyrosine kinases, platelet-derived and epidermal growth factor receptors. Engagement of the common downstream effector PI3K subsequently triggers activation of the MEK and mammalian target of rapamycin complex 2, which cooperate in regulating GLUT1 expression. Of note, inhibition of GLUT1 activity and/or expression is…

Bisphenol-A (BPA) is an environmentally ubiquitous estrogen-like endocrine-disrupting compound. Exposure to BPA in utero has been linked to female reproductive disorders, including endometrial hyperplasia and breast cancer. Estrogens are an etiological factor in many of these conditions. We sought to determine whether in utero exposure to BPA altered the global CpG methylation pattern of the uterine genome, subsequent gene expression, and estrogen response. Pregnant mice were exposed to an environmentally relevant dose of BPA or DMSO control. Uterine DNA and RNA were examined by using methylated DNA immunoprecipitation methylation microarray, expression microarray, and quantitative PCR. In utero BPA exposure altered the global CpG methylation profile of the uterine genome and subsequent gene expression. The effect on gene expression was not apparent until sexual maturation, which suggested that estrogen response was the primary alteration. Indeed, prenatal BPA exposure preferentially altered adult estrogen-responsive gene expression. Changes in estrogen response were accompanied by…

Early-life stress (ES) impairs cognition later in life. Because ES prevention is problematic, intervention is needed, yet the mechanisms that underlie ES remain largely unknown. So far, the role of early nutrition in brain programming has been largely ignored. Here, we demonstrate that essential 1-carbon metabolism-associated micronutrients (1-CMAMs; i.e., methionine and B vitamins) early in life are crucial in programming later cognition by ES. ES was induced in male C57Bl/6 mice from postnatal d (P)2-9. 1-CMAM levels were measured centrally and peripherally by using liquid chromatography-mass spectroscopy. Next, we supplemented the maternal diet with 1-CMAM only during the ES period and studied cognitive, neuroendocrine, neurogenic, transcriptional, and epigenetic changes in adult offspring. We demonstrate that ES specifically reduces methionine in offspring plasma and brain. Of note, dietary 1-CMAM enrichment during P2-9 restored methionine levels and rescued ES-induced adult cognitive impairments. Beneficial effects of this early dietary enrichment were associated with…

Aging and developmental programming are both associated with oxidative stress and endothelial dysfunction, suggesting common mechanistic origins. However, their interrelationship has been little explored. In a rodent model of programmed cardiovascular dysfunction we determined endothelial function and vascular telomere length in young (4 mo) and aged (15 mo) adult offspring of normoxic or hypoxic pregnancy with or without maternal antioxidant treatment. We show loss of endothelial function [maximal arterial relaxation to acetylcholine (71 ± 3 vs. 55 ± 3%) and increased vascular short telomere abundance (4.2-1.3 kb) 43.0 ± 1.5 vs. 55.1 ± 3.8%) in aged vs. young offspring of normoxic pregnancy (P < 0.05). Hypoxic pregnancy in young offspring accelerated endothelial dysfunction (maximal arterial relaxation to acetylcholine: 42 ± 1%, P < 0.05) but this was dissociated from increased vascular short telomere length abundance. Maternal allopurinol rescued maximal arterial relaxation to acetylcholine in aged offspring of normoxic or hypoxic pregnancy but not in young offspring of hypoxic…

The molecular mechanisms leading to and responsible for age-related, sporadic Alzheimer's disease (AD) remain largely unknown. It is well documented that aging patients with elevated levels of the amino acid metabolite homocysteine (Hcy) are at high risk of developing AD. We investigated the impact of Hcy on molecular clearance pathways in mammalian cells, including in vitro cultured induced pluripotent stem cell-derived forebrain neurons and in vivo neurons in mouse brains. Exposure to Hcy resulted in up-regulation of the mechanistic target of rapamycin complex 1 (mTORC1) activity, one of the major kinases in cells that is tightly linked to anabolic and catabolic pathways. Hcy is sensed by a constitutive protein complex composed of leucyl-tRNA-synthetase and folliculin, which regulates mTOR tethering to lysosomal membranes. In hyperhomocysteinemic human cells and cystathionine β-synthase-deficient mouse brains, we find an acute and chronic inhibition of the molecular clearance of protein products resulting in a buildup of abnormal proteins, including β-amyloid and phospho-Tau.…

The epithelial-mesenchymal transition (EMT), defined as transdifferentiation of epithelial cells into mesenchymal cells, is critical for embryonic development, wound healing, tissue regeneration, organ fibrosis, and cancer progression. Recently, the role of EMT in carcinogenesis has attracted much attention. Oncoviruses, including human papillomaviruses (HPVs), Epstein-Barr virus (EBV), and hepatitis B and C viruses (HBVs, HCVs), are known to be involved in the etiology of cancer and have been found to play important roles in cancer metastasis, especially in the EMT process. The HPV encoded oncoproteins E6 and E7 (E6/E7), EBV latent membrane protein-1 and -2A, EBV nuclear antigen, HBV-encoded X antigen, and nonstructural HCV protein 5A are all involved in the regulation of EMT. This review primarily focuses on the role of oncoviruses and their encoded proteins or signaling pathways in the EMT process. Understanding their roles will help us in the development of effective strategies for prevention and treatment of virus-related cancers.-Chen, X., Bode, A. M., Dong, Z., Cao, Y. The…

The ability to adapt cellular metabolism to nutrient availability is critical for survival. The liver plays a central role in the adaptation to starvation by switching from glucose-consuming processes and lipid synthesis to providing energy substrates like glucose to the organism. Here we report a previously unrecognized role of the tumor suppressor p53 in the physiologic adaptation to food withdrawal. We found that starvation robustly increases p53 protein in mouse liver. This induction was posttranscriptional and mediated by a hepatocyte-autonomous and AMP-activated protein kinase-dependent mechanism. p53 stabilization was required for the adaptive expression of genes involved in amino acid catabolism. Indeed, acute deletion of p53 in livers of adult mice impaired hepatic glycogen storage and induced steatosis. Upon food withdrawal, p53-deleted mice became hypoglycemic and showed defects in the starvation-associated utilization of hepatic amino acids. In summary, we provide novel evidence for a p53-dependent integration of acute changes of cellular energy status and the metabolic…

Although oat (1,3:1,4)-β-glucan (BG) has been shown to decrease blood cholesterol in intervention trials, the detailed mechanism is not yet defined, but restricted reabsorption of bile acids (BAs) has been hypothesized. Using pigs as a model for humans we demonstrated that, compared to the control, BG added to the diet for 26 d caused decreases of 24% in blood total BAs (TBAs), 34% in total cholesterol (TC), and 57% in LDL cholesterol (LDL-C) (P < 0.01); decreases of 20% TBA in the midjejunum and terminal ileum (P < 0.01); increases of 80% in cecal total neutral sterols (TNSs) including cholesterol (P < 0.01); a 50% reduction in BA active transport across ex vivo ileum after 40 min (P < 0.001); and 32% decrease in jejunal microvillus heights with apparent increased goblet cell activity. The results suggest that BG not only physically hinders the active reabsorption of BAs and uptake of cholesterol, but also changes the BAs profile with lower circulating levels without excess excretion in the feces, thus resulting in reduced blood TC and LDL-C. Fermentation of sterols reaching the…

Neutrophil [polymorphonuclear leukocyte (PMN)] transepithelial migration (TEM) is a hallmark of inflammatory mucosal disorders. PMN TEM is associated with epithelial injury; however, mechanisms involved in this process are not well defined. The current work describes a new mechanism whereby deposition of PMN membrane-derived microparticles (PMN-MPs) onto intestinal epithelial cells (IECs) during TEM leads to loss of epithelial cadherins, thus promoting epithelial injury and increased PMN recruitment. PMN-MPs secreted by activated PMNs during TEM displayed a high level of enzymatically active matrix metalloproteinase 9 (MMP-9), and were capable of mediating potent effects on IEC integrity. Isolated PMN-MPs efficiently bound to IEC monolayers and induced cleavage of desmoglein-2 (DSG-2) but not E-cadherin, leading to disruption of IEC intercellular adhesions. Furthermore, PMN-MP binding to intestinal epithelium in vitro in transwell assays and in vivo in ligated intestinal loop preparations facilitated increases in PMN TEM. These effects were MMP-9 dependent and were reversed in the…

Previous rodent studies have shown that maternal voluntary exercise during pregnancy leads to metabolic changes in adult offspring. We set out to test whether maternal voluntary exercise during pregnancy also induces persistent changes in voluntary physical activity in the offspring. Adult C57BL/6J female mice were randomly assigned to be caged with an unlocked (U) or locked (L) running wheel before and during pregnancy. Maternal running behavior was monitored during pregnancy, and body weight, body composition, food intake, energy expenditure, total cage activity, and running wheel activity were measured in the offspring at various ages. U offspring were slightly heavier at birth, but no group differences in body weight or composition were observed at later ages (when mice were caged without access to running wheels). Consistent with our hypothesis, U offspring were more physically active as adults. This effect was observed earlier in female offspring (at sexual maturation). Remarkably, at 300 d of age, U females achieved greater fat loss in response to a 3-wk voluntary exercise…

ABSTRACT As many as 10% of bone fractures heal poorly, and large bone defects resulting fromtrauma, tumor, or infection may not heal without surgical intervention. Activation of adenosine A 2A receptors (A 2A Rs) stimulates bone formation. Ticagrelor and dipyridamole inhibit platelet function by inhibiting P2Y 12 receptors and platelet phosphodiesterase, respectively, but share the capacity to inhibit cellularuptake of adenosine and thereby increase extracellular adenosine levels. Because dipyridamole promotes bone regeneration by an A 2A R‐mediated mechanism we determined whether ticagrelor could regulate the cells involved in bone homeostasis and regeneration in a murine model and whether inhibition of P2Y 12 or indirect A 2A R activation via adenosine was involved. Ticagrelor, dipyridamole and the active metabolite of clopidogrel (CAM), an alternative P2Y 12 antagonist, inhibited osteoclast differentiation and promoted osteoblast differentiation in vitro . A 2A R blockade abrogated the effects of ticagrelor and dipyridamole on osteoclast and osteoblast differentiation where as A…

Calcium- and integrin-binding protein 1 (CIB1) is a small, ubiquitously expressed protein that was first identified as an intracellular binding partner of a platelet-specific α-integrin cytoplasmic tail. Although early studies revealed a role for CIB1 in regulating platelet integrin activity, recent studies have indicated a more diverse role for CIB1 in many different cell types and processes, including calcium signaling, migration, adhesion, proliferation, and survival. Increasing evidence also points to a novel role for CIB1 in cancer and cardiovascular disease. In addition, an array of CIB1 binding partners has been identified that provide important insight into how CIB1 may regulate these processes. Some of these binding partners include the serine/threonine kinases, p21-activated kinase 1 (PAK1), apoptosis signal-regulating kinase 1 (ASK1), and polo-like kinase 3 (PLK3). Structural and mutational studies indicate that CIB1 binds most or all of its partners via a well-defined hydrophobic cleft. Although CIB1 itself lacks known enzymatic activity, it supports the PI3K/AKT and…

The role of peroxisome proliferator-activated receptor α (PPARα)-mediated metabolic remodeling in cardiac adaptation to hypoxia has yet to be defined. Here, mice were housed in hypoxia for 3 wk before in vivo contractile function was measured using cine MRI. In isolated, perfused hearts, energetics were measured using (31)P magnetic resonance spectroscopy (MRS), and glycolysis and fatty acid oxidation were measured using [(3)H] labeling. Compared with a normoxic, chow-fed control mouse heart, hypoxia decreased PPARα expression, fatty acid oxidation, and mitochondrial uncoupling protein 3 (UCP3) levels, while increasing glycolysis, all of which served to maintain normal ATP concentrations ([ATP]) and thereby, ejection fractions. A high-fat diet increased cardiac PPARα expression, fatty acid oxidation, and UCP3 levels with decreased glycolysis. Hypoxia was unable to alter the high PPARα expression or reverse the metabolic changes caused by the high-fat diet, with the result that [ATP] and contractile function decreased significantly. The adaptive metabolic changes caused by hypoxia in…

Nonalcoholic fatty liver disease (NAFLD) is widespread in adults and children. Early exposure to maternal obesity or Western‐style diet (WD) increases steatosis and oxidative stress in fetal liver and is associated with lifetime disease risk in the offspring. Pyrroloquinoline quinone (PQQ) is a natural antioxidant found in soil, enriched in human breast milk, and essential for development in mammals. We investigated whether a supplemental dose of PQQ, provided prenatally in a mouse model of diet‐induced obesity during pregnancy, could protect obese offspring from progression of NAFLD. PQQ treatment given pre‐ and postnatally in WD‐fed offspring had no effect on weight gain but increased metabolic flexibility while reducing body fat and liver lipids, compared with untreated obese offspring. Indices of NAFLD, including hepatic ceramide levels, oxidative stress, and expression of proinflammatory genes ( Nos2 , Nlrp3 , Il6 , and Ptgs2 ), were decreased in WD PQQ‐fed mice, concomitant with increased expression of fatty acid oxidation genes and decreased Pparg expression. Notably, these…

Apelin, a (neuro)vasoactive peptide, plays a prominent role in controlling cardiovascular functions and water balance. Because the in vivo apelin half-life is in the minute range, we aimed to identify metabolically stable apelin-17 (K17F) analogs. We generated P92 by classic chemical substitutions and LIT01-196 by original addition of a fluorocarbon chain to the N terminus of K17F. Both analogs were much more stable in plasma (half-life >24 h for LIT01-196) than K17F (4.6 min). Analogs displayed a subnanomolar affinity for the apelin receptor and behaved as full agonists with regard to cAMP production, ERK phosphorylation, and apelin receptor internalization. Ex vivo, these compounds induced vasorelaxation of rat aortas and glomerular arterioles, respectively, precontracted with norepinephrine and angiotensin II, and increased cardiac contractility. In vivo, after intracerebroventricular administration in water-deprived mice, P92 and LIT01-196 were 6 and 160 times, respectively, more efficient at inhibiting systemic vasopressin release than K17F. Administered intravenously (nmol/kg…

ABSTRACT In this study, we examined the functional importance of EZH2 during skeletal development and homeostasis using the conditional deletion of Ezh2 ( Ezh2 fl/fl ) in early mesenchyme with the use of a Prrx‐1‐cre driver mouse ( Ezh2 +/+ ). Heterozygous ( Ezh2 +/− ) newborn and 4‐wk‐old mice exhibited increased skeletal size, growth plate size, and weight when compared to the wild‐type control ( Ezh2 +/+ ), whereas homozygous deletion of Ezh2 ( Ezh2 −/− )resultedin skeletal deformities and reduced skeletal size, growth plate size, and weight in newborn and 4‐wk‐old mice. Ezh2 −/− mice exhibited enhanced trabecular patterning. Osteogenic cortical and trabecular bone formation was enhanced in Ezh2 +/ − and Ezh2 −/− animals. Ezh2 +/ − and Ezh2 −/− mice displayed thinner cortical bone and decreased mechanical strength compared to the wild‐type control. Differences in cortical bone thickness were attributed to an increased number of osteoclasts, corresponding with elevated levels of the bone turnover markers cross‐linked C‐telopeptide‐1 and tartrate‐resistant acid phosphatase,…

Reactive oxygen species (ROS) play a role in intracellular signaling during osteoclastogenesis. We previously reported that transcriptional factor nuclear factor E2-related factor 2 (Nrf2) was exported from the nucleus to the cytoplasm by receptor activator of nuclear factor-κB ligand (RANKL), and that Nrf2 negatively regulated osteoclastogenesis via antioxidant enzyme up-regulation. Knockout mice of BTB and CNC homology 1 (Bach1)-the competitor for Nrf2 in transcriptional regulation-was known to attenuate RANKL-mediated osteoclastogenesis, although the mechanism remains unclear. Therefore, we hypothesized that RANKL could be involved in the nuclear translocation of Bach1, which would attenuate Nrf2-mediated antioxidant enzymes, thereby augmenting intracellular ROS signaling in osteoclasts. RANKL induced Bach1 nuclear import and Nrf2 nuclear export. Induction of Bach1 nuclear export increased Nrf2 nuclear import, augmented antioxidant enzyme expression, and, thus, diminished RANKL-mediated osteoclastogenesis via attenuated intracellular ROS signaling. Finally, an in vivo mouse bone…

Tumor exosomes are emerging as antitumor immunity regulators; however, their effects on secondary exosome secretion by distal organs have not been explored. We have previously demonstrated that suppression of exosomes at the distal tumor site of pancreatic ductal adenocarcinoma (PDAC) ablated the development of salivary biomarker profile. Here, we explore the function of salivary exosomes from tumor-bearing mice in immune surveillance. We provide evidence that salivary exosomes from mice with PDAC exhibit a suppressive effect that results in reduced tumor-killing capacity by NK cells. Salivary exosomes from mice with PDAC where pancreatic tumors were engineered to suppress exosome biogenesis failed to suppress NK cell cytotoxic potential against tumor cells, as opposed to salivary exosomes from mice with PDAC with normal tumor exosome biogenesis. These results reveal an important and previously unknown mechanism of antitumor immune regulation and provide new insights into our understanding of the alterations of this biofluid during tumor development.-Katsiougiannis, S., Chia, D.,…

Most satiety-inducing obesity therapeutics, despite modest efficacy, have safety concerns that underscore the need for effective peripherally acting drugs. An attractive therapeutic approach for obesity is to optimize/maximize energy expenditure by increasing energy-utilizing thermogenic brown adipose tissue. We used in vivo and in vitro models to determine the role of estrogen receptor β (ER-β) and its ligands on adipose biology. RNA sequencing and metabolomics were used to determine the mechanism of action of ER-β and its ligands. Estrogen receptor β (ER-β) and its selective ligand reprogrammed preadipocytes and precursor stem cells into brown adipose tissue and increased mitochondrial respiration. An ER-β-selective ligand increased markers of tricarboxylic acid-dependent and -independent energy biogenesis and oxygen consumption in mice without a concomitant increase in physical activity or food consumption, all culminating in significantly reduced weight gain and adiposity. The antiobesity effects of ER-β ligand were not observed in ER-β-knockout mice. Serum metabolite profiles…

Clinical trials have shown that administration of the nematode Trichuris suis can be beneficial in treating various immune disorders. To provide insight into the mechanisms by which this worm suppresses inflammatory responses, an active component was purified from T. suis soluble products (TsSPs) that suppress---- TNF and IL-12 secretion from LPS-activated human dendritic cells (DCs). Analysis by liquid chromatography tandem mass spectrometry identified this compound as prostaglandin (PG)E2. The purified compound showed similar properties compared with TsSPs and commercial PGE2 in modulating LPS-induced expression of many cytokines and chemokines and in modulating Rab7B and P2RX7 expression in human DCs. Furthermore, the TsSP-induced reduction of TNF secretion from DCs is reversed by receptor antagonists for EP2 and EP4, indicating PGE2 action. T. suis secretes extremely high amounts of PGE2 (45-90 ng/mg protein) within their excretory/secretory products but few related lipid mediators as established by metabololipidomic analysis. Culture of T. suis with several cyclooxygenase (COX)…

The NLRP3 inflammasome is a multimeric protein complex that controls the production of IL-1β, a cytokine that influences the development of both innate and adaptive immune responses. Helminth parasites secrete molecules that interact with innate immune cells, modulating their activity to ultimately determine the phenotype of differentiated T cells, thus creating an immune environment that is conducive to sustaining chronic infection. We show that one of these molecules, FhHDM-1, a cathelicidin-like peptide secreted by the helminth parasite, Fasciola hepatica, inhibits the activation of the NLRP3 inflammasome resulting in reduced secretion of IL-1β by macrophages. FhHDM-1 had no effect on the synthesis of pro-IL-1β. Rather, the inhibitory effect was associated with the capacity of the peptide to prevent acidification of the endolysosome. The activation of cathepsin B protease by lysosomal destabilization was prevented in FhHDM-1-treated macrophages. By contrast, peptide derivatives of FhHDM-1 that did not alter the lysosomal pH did not inhibit secretion of IL-1β. We propose a novel…

Exosomes are a class of cell-released small vesicles that mediate intercellular communication by delivering functional factors to recipient cells. During hepatitis C virus (HCV) infection, the interaction between liver resident macrophages and hepatocytes is a key component in liver innate immunity. In this study, we explored the role of exosomes in the delivery of innate anti-HCV factors to hepatocytes from macrophages. We showed that supernatant from TLR3-activated macrophage cultures could efficiently inhibit HCV replication in Huh7 cells. This macrophage-mediated anti-HCV activity was through exosomes because inhibiting exosomes could abrogate the action of macrophages. Further analyses demonstrated that TLR3-activated macrophages release exosomes that contain anti-HCV microRNA (miRNA)-29 family members. Inhibiting miRNA29 could restore HCV replication. These findings suggest a novel antiviral mechanism in liver innate immunity against HCV infection and provide insights to support further studies on developing exosome-based delivery system for disease treatment.-Zhou, Y., Wang,…

IFN regulatory factor 1 (IRF1) is one of the most important IFN-stimulated genes (ISGs) in cellular antiviral immunity. Although hepatitis E virus (HEV) is a leading cause of acute hepatitis worldwide, how ISGs counteract HEV infection is largely unknown. This study was conducted to investigate the effect of IRF1 on HEV replication. Multiple cell lines were used in 2 models that harbor HEV. In different HEV cell culture systems, IRF1 effectively inhibited HEV replication. IRF1 did not trigger IFN production, and chromatin immunoprecipitation sequencing data analysis revealed that IRF1 bound to the promoter region of signal transducers and activators of transcription 1 (STAT1). Functional assay confirmed that IRF1 could drive the transcription of STAT1, resulting in elevation of total and phosphorylated STAT1 proteins and further activating the transcription of a panel of downstream antiviral ISGs. By pharmacological inhibitors and RNAi-mediated gene-silencing approaches, we revealed that antiviral function of IRF1 is dependent on the JAK-STAT cascade. Furthermore, induction of ISGs…

ABSTRACT Pulmonary fibrosis is a progressive and often fatal condition that is believed to be partially orchestrated by macrophages. Mechanisms that control migration of these cells into and within the lung remain undefined. We evaluated thecontributions of the semaphorinreceptor, plexinC1 (PLXNC1), andtheexocytic calcium sensor, synaptotagmin 7 (Syt7), in these processes. Weevaluated the role of PLXNC1 in macrophagemigration by using Boyden chambers and scratch tests, characterized its contribution to experimentally induced lung fibrosis inmice, and defined themechanism for our observations. Our findings reveal that relative to controlparticipants, patients with idiopathic pulmonary fibrosis demonstrate excessive monocyte migration and underexpression of PLXNC1 in the lungs and circulation, a finding that is recapitulated in the setting of scleroderma‐related interstitial lung disease. Relative to wild type, PLXNC1 ‐/‐ mouse macrophages are excessively migratory, and PLXNC1 ‐/‐ mice show exacerbated collagen accumulation in response to either inhaled bleomycin or inducible lung…

We studied the physiologic roles of the hyaluronidase (HYAL) 1 and HYAL2 in hyaluronan (HA) turnover. HA was localized and quantified using HA binding proteins in various tissues of Hyal1(-/-) and Hyal2(-/-) mice (knockout mice) as well as control mice. HA MW was determined using gel filtration chromatography. HA endocytosis in liver nonparenchymal cells (NPCs) was quantified in vivo Both Hyal1 and Hyal2 knockout mice showed HA accumulation in peripheral tissues without changes in HA MW distribution. HYAL2 deficiency induced buildup of very high MW (>3.10(6) Da) HA in lymph and serum with severe lymph node distortion. The lack of HYAL2 also impaired high MW HA endocytosis by liver NPCs. HYAL1 deficiency led to a moderate increase in serum HA concentration without changes in HA MW distribution and to HA overload of liver NPCs. Wild-type C57BL/6 mice served as controls. In HA injection experiments, saline-injected mice served as additional controls. We conclude that: 1) HYAL1 and HYAL2 are both needed for tissue HA catabolism; 2) HYAL2 is required for high MW HA clearance in lymph…

Adiponectin is an adipocyte-derived adipokine with potent antidiabetic, anti-inflammatory, and antiatherogenic activity. Long-term, high-fat diet results in gain of body weight, adiposity, further inflammatory-based cardiovascular diseases, and reduced adiponectin secretion. Vitamin A derivatives/retinoids are involved in several of these processes, which mainly take place in white adipose tissue (WAT). In this study, we examined adiponectin expression as a function of dietary high-fat and high-vitamin A conditions in mice. A decrease of adiponectin expression in addition to an up-regulation of aldehyde dehydrogenase A1 (ALDH1A1), retinoid signaling, and retinoic acid response element signaling was selectively observed in WAT of mice fed a normal-vitamin A, high-fat diet. Reduced adiponectin expression in WAT was also observed in mice fed a high-vitamin A diet. Adipocyte cell culture revealed that endogenous and synthetic retinoic acid receptor (RAR)α- and RARγ-selective agonists, as well as a synthetic retinoid X receptor agonist, efficiently reduced adiponectin expression, whereas…