In this review, we will summarize recent developments in innovative and powerful molecular approaches, including knockout mouse models, transgenic mouse models, and astrocyte-targeted gene transfer/expression, which have led to advances in understanding astrocyte biology in vivo that were heretofore inaccessible to experimentation. We will examine the recently improved understanding of the roles of astrocytes – with an emphasis on astrocyte signaling – in the context of both the healthy and diseased brain, discuss areas where the role of astrocytes remains debated, and suggest new research directions.Astrocytes are a predominant glial cell type in the nervous systems, and are becoming recognized as important mediators of normal brain function as well as neurodevelopmental, neurological, and neurodegenerative brain diseases. Although numerous potential mechanisms have been proposed to explain the role of astrocytes in the normal and diseased brain, research into the physiological relevance of these mechanisms  In particular, we will describe the main molecular approaches used in this field, including knockout (KO) mouse models, transgenic mouse models, and astrocyte-targeted gene transfer and expression using adeno-associated viral (AAV) or in utero electroporation (IUE) approaches. We will also touch on a remarkable recent study involving engraftment of genetically modified human glial progenitors into the mouse brain, providing insight into the role of human astrocytes in the unique cognitive abilities of the human brain. The present review does not attempt to be comprehensive; rather it highlights certain major themes and areas of recent progress on the roles of astrocytes in brain function, with an emphasis on astrocyte signaling and in vivo studies. For complementary reviews on astrocyte function in health and disease with an emphasis on molecular approaches, readers are directed to . This results in the production of a mutated or truncated protein or, more often, no protein at all. ES cells that do not take up the foreign construct are killed through exposure to neomycin, and those that have successfully replaced the gene or the exons of this gene survive and are subsequently microinjected into mouse blastocysts, which are then grown in surrogate mouse uteri. Strategic mating of the chimeric mice will ultimately result in a mouse with the gene globally eliminated .As research into astroglia physiology One of the first genes that was removed in astrocytes is the gene encoding glial fibrillary acidic protein GFAP; . GFAP isLacZ cassette  and Purkinje cells during induction and maintenance of cerebellar LTD in vivo  CNS mechanical trauma  cel trauma , (iii) kl trauma , and  the heterogeneity of astrocyte morphology and physiology  thysiology ; (iii) tysiology ; (iv) thysiology ; and (v)ysiology . Suitablysiology . An alteysiology . Isoform3R2). Two constitutive IP3R2-deficient mouse models have been obtained through a variation of the gene targeting strategy. In one, the targeting construct was generated by flanking exon 3 of IP3R2 with two LoxP sites and flanking the neo-cassette by FRT sites  to quickly modulate synaptic transmission and plasticity , or short- and long-term plasticity (NMDA receptor-dependent LTP) in the hippocampus ex vivo  have distinct effects on LTP , which is complicated because most of the IP3R1-deficient mice die in utero  astrocytic Ca2+ dynamics are linked to the symptoms of Alzheimer’s disease . The site-specific recombination system of the P1 bacteriophage using Cre and LoxP-flanked genes is well documented , and a second that has been made via homologous recombination in ES cells expressing the 34-base-pair LoxP site (recognition site for Cre recombinase) flanking the gene of interest or one or more exons of this gene. When these two mouse lines are crossed, the gene or exons of interest are excised to obtain the desired cKO mouse line . While cKO mice are very valuable tools, their use has been limited in the astrocyte field because the promoters that are known to be astrocyte specific in the adult are also expressed in progenitor cells of the developing brain. As a consequence, not only astrocytes, but also a large percentage of neurons and oligodendrocytes, exhibit recombination when using astrocyte-specific promoters to drive the expression of Cre recombinase . To this end, mice have been developed which express Cre recombinase fused to a mutated form of the human estrogen receptor (ERT2) that restricts the fusion protein to the cytoplasm unless exposed to the estrogen analog tamoxifen  allows study of the effect of gene deletion specifically in astrocytes if tamoxifen is given to mice at later development stages . One limitation of the CreERT2 inducible system and inducible systems in general is that recombination efficiency may be low and therefore the phenotypes generated may be more subtle, as has been reported for Cx43 compared to floxed reporter genes  expression in astrocytes (LoxP-flanked CB1R (T2 under the control of the human GFAP promoter (1R-KO) has been used to investigate the mechanisms underlying impairment of working memory, which is one of the most important deleterious effects of marijuana intoxication in humans (1R (GPCR predominantly coupling to Gi proteins) is highly abundant in the CNS  the apparent mechanistic discrepancy between this in vivo cannabinoid-induced LTD and previous ex vivo “gliotransmission” studies suggesting that astrocytic CB1R-mediated Ca2+ increases trigger the release of glutamate to activate pre-synaptic mGluRs that leads to glutamate presynaptic release and subsequent potentiation of postsynaptic NMDA receptor-mediated currents  tysiology ; (iv) th+ uptake ; and (v)+ uptake . Furtheri  astrocytic Gi signaling leads to distinct responses depending on the area of the brain ; or (iii) astrocytic Gi GPCRs are involved in different functions depending on the developmental stage (mature vs. immature astrocytes). Additionally, another possibility that has not been addressed thoroughly in the field when investigating the role of astrocyte signaling in general is that different astrocytic Gi GPCRs may activate distinct signaling pathways even though they are known to couple to Gi proteins. In other words, depending on the endogenous or synthetic agonist used to active a specific Gi GPCR, this receptor may show a different bias toward other G proteins over Gi or different functional selectivity for β-arrestin recruitment over Gi activation  in physiology and pathology likely reflect the complex interactions of multiple signaling pathways and provides an explanation for seemingly contradictory observations. Examples of this have already been observed relative to astrocyte Ca2+ signaling which is traditionally ascribed to Gq GPCR signaling pathways, yet astrocytic Gi-coupled GABAB and CB1Rs also have been reported to evoke Ca2+ elevations in astrocytes  expression of a genetically modified gene (transgene); (ii) reversible regulation of an endogenous gene; (iii) viral transduction of genes; or (iv) in utero gene electroporation (IUE).As methods for generating full or inducible conditional gene KO have been useful in deciphering some of the astrocyte gene functions Figure 2A) and inducible  transgenesis allowing for astrocyte-specific expression of transgenes, constitutively or at specific times, have been used in the field. These approaches employ a small transcriptional unit derived from astrocyte-specific promoters. Of importance for the topic of this review, mapping of the transcriptional regulatory elements of the GFAP promoter has been critical to develop regulatory units (promoters) of small size to direct transgene expression in the majority of astrocytes in vivo without significant expression in other cell types in the brain . The resulting pups are identified as transgenic by polymerase chain reaction and checked for astrocyte-specific transgene expression. This constitutive gain-of-function approach however does not provide temporal control of the transgene expression, which can be problematic when studying the function of a transgene during developmental stages as mentioned above . This system is bi-transgenic, which means that it involves the mating of two different transgenic mouse lines in order to produce a new tetracycline-regulated transgenic mouse line designed to activate the expression of a transgene in a specific cell type at a specific time point. The first line contains an astrocyte-specific promoter driving the expression of the tetracycline (tet) transactivator (tTA). The second line carries the transgene of interest driven by the tet operon (TetO) DNA-binding element fused to a minimal promoter from cytomegalovirus. In this system, bigenic mice are maintained on tetracycline (doxycycline) to block transgene expression. When doxycycline is removed at a given time, tTA then binds to the TetO minimal promoter leading to targeted expression of the transgene of interest .Conventional  receptors by the applied agonist in addition to the intended astrocytic targets. As a consequence, it has been difficult to determine the effect of selectively stimulating astrocytic Gq GPCR-mediated Ca2+ signaling cascades on physiological processes such as synaptic transmission and plasticity. This complication has led investigators to consider previous reports of gliotransmission with caution.One important limitation in addressing the role of astrocytic GPCR signaling in neurophysiology has been the inability to pharmacologically activate astrocytic GPCRs in a cell type-specific manner. Indeed, astrocytes cascades  that areq GPCR Ca2+ signaling. In order to overcome some of the limitations associated with traditional pharmacological approaches, two novel transgenic mouse models were developed . In one transgenic mouse line the green fluorescent protein (GFP)-tagged MrgA1 receptor is transcribed from the TetO promoter. When crossed with a second transgenic line in which tTA is targeted to astrocytes using the human GFAP promoter, a bigenic line (referred to as the MrgA1 mice) is obtained, in which MgrA1-GFP is selectively expressed in the vast majority of astrocytes in the absence of doxycycline . To overcome some of these obstacles, a new GFAP-hM3Dq mouse line was created  that does not respond to endogenous ligands, but instead responds to an inert synthetic ligand  that crosses the BBB and activates signaling cascades in a similar fashion as endogenous Gq GPCRs . Inducible and reversible expression of the transgene is not a constraint when using the DREADD technology, as hM3Dq can be activated postdevelopmentally through intraperitoneal CNO injection. Immunohistochemical screening in adult mice indicates that expression of hM3Dq is restricted to astrocytes within the CNS, and in non-myelinating Schwann cells of sympathetic, sensory, and sciatic nerves, as well as satellite cells in sympathethic, parasympathetic, and sensory ganglia within the PNS. Such expression is expected for a GFAP-driven transgene and allows CNO-induced global stimulation in GFAP+ glial cells within the CNS and PNS. This provides a powerful approach, as it can reveal most of the physiological and behavioral phenotypes mediated by GFAP+ glial cell Gq GPCR signaling. Acute CNO intraperitoneal injections of GFAP-hM3Dq mice resulted in previously unreported and long-lasting (minutes to hours) modulation of autonomic nervous system (ANS) function, including increased heart rate, blood pressure, and saliva formation, as well as decreased body temperature. Furthermore, changes in activity-related behavior and motor coordination were observed in CNO-treated GFAP-hM3Dq mice. To address whether ANS and activity-related effects were due to Ca2+ or other signaling pathways downstream of Gq GPCR activation, the GFAP-hM3Dq mice were crossed with IP3R2 KO mice in order to generate GFAP-hM3Dq mice in which IP3R2-dependent Ca2+ increases were abolished. Interestingly, CNO-treated GFAP-hM3Dq/IP3R2-deficient mice exhibited similar autonomic and motor modulation as CNO-treated GFAP-hM3Dq mice. These findings suggest that CNO-induced phenotypes are not dependent on IP3R2-dependent Ca2+ increases. Thus, other (non-Ca2+) signaling molecules activated by Gq GPCRs in GFAP+ glial cells may be important contributors to the functional effects of CNO. Collectively, these findings open new avenues into investigation of GFAP+ glial cell  function in animal physiology. Further studies employing local CNO infusion into specific regions of the CNS or PNS, and specific blockers of Gq GPCR signaling molecules , will help dissect out the areas of the nervous system and the signaling pathways that are responsible for each of the CNO-induced phenotypes. Viral transduction of hM3Dq in wild-type mice is also a potential strategy to help determine what areas of the CNS vs. PNS are involved in the effects observed in CNO-treated GFAP-hM3Dq mice. Finally, when specific markers of different subpopulations of CNS astrocytes become available, it will be possible to use new promoters to create transgenic mice expressing hM3Dq in distinct subsets of astrocytes. This will allow determination of whether a specific subset of astrocytes is responsible for all of the phenotypes observed in CNO-treated GFAP-hM3Dq mice, or whether different subsets of astrocytes are responsible for distinct phenotypes. Clearly, there is much to be learned with regard to the role of astrocytes, and more generally of GFAP+ glial cells, in complex physiology and behavior. Collectively, the findings of this study point to astrocytes as potential therapeutic targets for some ANS or motor dysfunctions.More recently, another transgenic mouse line has been developed in order to facilitate investigation of the role of astrocytic G in vivo . One of  created . The innGq GPCRs . Such neGq GPCRs , and is Gq GPCRs . This moLoxP-flanked selectable neo marker and transcriptional/translational stop cassette  located in the endogenous gene of interest to generate a null mutant that can be activated by Cre-mediated recombination. This construct is placed into a targeting vector, which is inserted into ES cells and introduced into the mouse genome through homologous recombination  to eventually generate a mouse line in which expression of the targeted endogenous gene is suppressed. When this Cre responding mouse line is crossed with a transgenic line expressing Cre recombinase under a cell type-specific promoter, recombination of LoxP sites excises the neostop cassette, thus re-activating expression of the endogenous gene conditionally  and with temporal control in a specific cell population . This approach is powerful in the sense that it enables study of important questions regarding the phenotype reversibility of certain diseases involving the loss of a single gene function, and thus has the potential to open doors for future therapeutic approaches , (lox-Stop::hGFAP–CreERT2) was thus specifically designed for the investigation of astrocytic function in RTT  and molecular underpinnings of RTT is likely to contribute to the understanding of the pathogenesis of a broader class of autism spectrum disorders. Finally, the use of conditional endogenous gene repair mutations has a clear application for studying astrocytic contributions to other single gene diseases.Rett’s syndrome (RTT) is an X-chromosome-linked autism spectrum disorder caused by the loss of function of the epigenetic factor methyl-CpG-binding protein 2 MeCP2; . MeCP2 aal MeCP2  using dial MeCP2 . Howeveral MeCP2 . In partfunction . Global function . Based oreERT2), , the resn in RTT . The MeCications . Deciphe2+ activity.A new technique that is gaining momentum in the study of astrocyte function is viral-mediated delivery of transgenic constructs to express or perturb a protein or molecule of interest. This strategy offers an attractive alternative to generation of transgenic mouse lines, which can be very time consuming and expensive due to costs associated with maintenance of animal lines and genotyping. Viral mediated gene delivery is reviewed in detail in this special issue  and therBriefly, in these experiments, a transgenic construct is inserted into a recombinant AAV vector which is then injected into the brain of wild-type mice. Approximately 2 weeks is sufficient for the viral vector to be incorporated into the host genome and for high expression of the transgenic construct to occur . Several2+ fluctuations in astrocytes. Monitoring astrocyte Ca2+ activity continues to be a primary technique in the study of astrocytes ever since it was discovered in vitro that astrocyte Ca2+ elevations modulate neuronal excitability  overcomes many of the limitations associated with the traditional approaches described above. It can be thought of as “high resolution bulk-loading” in that many astrocytes can be monitored at once and with the excellent signal-to-noise offered by the latest GCaMPs. Because the GECIs are delivered using an astrocyte-specific promoter, no secondary labeling is necessary to confirm the cells as astrocytes. Studies have indicated very specific transfection of astrocytes using this technique . Recentlrotocols , but beceaflets” . Becausedynamics  in astro2+ microdomains and conditions under which Ca2+ elevations occur, high levels of expression may result in significant calcium buffering with unintended consequences. Perhaps a more significant concern associated with the use of AAV vectors is the possibility of inducing reactive gliosis. Astrocytic Ca2+ signaling is disrupted in reactive astrocytes, with typically exaggerated and more widely propagating Ca2+ elevations and with increased propensity for gliotransmitter release  obtained by magnetic-assisted cell sorting using antibodies against PSA-NCAM and A5B5 cell surface antigens. The A2B5+/PSA-NCAM- glial cells were expanded via a cell cultured protocol that promoted differentiation into astrocytes. Prior to transplantation into brains of neonatal immune-deficient mice, GPCs were transduced using a VSVg-pseudotyped lentiviral-CMC-EGFP in order to label them with a GFP. Once transplanted, mice matured to become adult chimeras for both mouse and human astrocytes  on astrocyte protein expression and function in development and disease. Finally, there has been a strong tendency to focus on the immediate, short-term consequences of manipulating astrocyte receptors and signaling molecules on neuronal activity and brain function. Based on the predominance of metabotropic signaling cascades in astrocytes and the role of astrocytes in homeostasis of the synaptic microenvironment, the role of astrocyte-to-neuron communication may be tuned toward long-term regulatory functions. Therefore, another area of future study is to examine the outcome of stimulating astrocytic signaling cascades on gene transcription, synthesis of new proteins, and long-term regulation of cellular processes in neurons, astrocytes, and other glial cell types.Important advances continue to be made in development of molecular tools to understand astrocyte function and the effect of manipulating individual astrocytic signaling molecules on neuronal activity and pathophysiology of the brain The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In the past, peritoneal metastases (PM) were considered as a final stage of cancer, and patients were offered a palliative chemotherapy or at best supportive care. In the early 1990s, some surgeons developed a new therapeutic alternative based on the combined treatment. In this curative intent, the macroscopic disease was treated with cytoreductive surgery (CRS) followed by treating residual microscopic disease with a direct intraabdominal application of intraperitoneal chemotherapy using peroperative hyperthermic intraperitoneal chemotherapy (HIPEC) and/or under normothermia of early postoperative intraperitoneal chemotherapy (EPIC). In 2003, prolonged survival of patients affected by peritoneal metastases of colorectal origin with complete cytoreduction followed by HIPEC was reported in phase III prospective-randomized trial [Besides these improvements, the long-term outcome of these patients is still not satisfied. Further studies need to be conducted with pharmacokinetics of chemotherapeutics and molecular biology studies to develop new therapeutic approaches in this comprehensive strategy.This special issue of peritoneal metastases offers 18 papers, which consists of 3 papers about pharmacokinetics during intrapertieoneal chemotherapy, 3 about new methods of chemotherapy, 2 about mechanisms and treatment of pulmonary/pleural metastases from peritoneal metastases of appendiceal neoplasm, 3 about new surgical methods and concept, 3 about prevention of recurrence after CC-0 resection, 2 about laparoscopic diagnosis and treatment, and 2 about postoperative morbidity and mortality. These topics covered point to new directions in peritoneal metastases due to gastric, colorectal, appendiceal, and ovarian cancer.Based on the present results, we have to continue our research and interventions to conquer this refractory disease.I have to express my deep gratitude to all the guest editors and expert colleagues all over the world who contributed to the special issue.Yutaka YonemuraYutaka YonemuraYan LiYan LiPaul H. SugarbakerPaul H. SugarbakerPompiliu PisoPompiliu Piso


The study by Tam et al  revealedU, α < .05) decrease in viral load on day 1 of dexamethasone treatment, concomitant with decreased, but still elevated, levels of select inflammatory cytokines . By day 3, suppression of cytokines was still evident, but viral titers had rebounded to the same level as in untreated samples. Viral titers for dexamethasone-treated samples did not exceed those in untreated samples on day 3 or day 5, presumably because the inflammatory response remained sufficiently intact to prevent enhanced viral load.We investigated dexamethasone as a dengue therapeutic in primary monocyte-derived macrophages from dengue-naive donors. From these samples, we collected cytokine and viral titer measurements at days 1, 3, and 5 and note an interesting phenomenon that we believe complements the results obtained in patients . Although these drugs are capable of reducing inflammatory mediators in important targets of infection in tissues (macrophages), the systemic effects and related severe pathologies may not be affected. Alternatively, the effectiveness of treatment may depend critically on the time of drug administration. Immediate treatment following infection may be able to help direct the developing immune response to effective viral clearance while preventing severe symptoms in the subset of patients at risk of shock or hemorrhage; however, this is an unrealistic option in the clinical setting. We suggest that modest immunosuppression may be safe for use in these diseases as an adjunctive therapy in coordination with a direct-acting antiviral capable of reducing pathogen load efficiently. Future experiments investigating such a strategy may unlock the therapeutic potential of otherwise nonviable options.


Non-alcoholic fatty liver disease (NAFLD), the hepatic manifestation of the metabolic syndrome, is a complex multifactorial disease characterized by metabolic deregulations that include accumulation of lipids in the liver, lipotoxicity, and insulin resistance. The progression of NAFLD to non-alcoholic steatohepatitis and cirrhosis, and ultimately to carcinomas, is governed by interplay of pro-inflammatory pathways, oxidative stress, as well as fibrogenic and apoptotic cues. As the liver is the major organ of biotransformation, deregulations in hepatic signaling pathways have effects on both, xenobiotic and endobiotic metabolism. Several major nuclear receptors involved in the transcription and regulation of phase I and II drug metabolizing enzymes and transporters also have endobiotic ligands including several lipids. Hence, hepatic lipid accumulation in steatosis and NAFLD, which leads to deregulated activation patterns of nuclear receptors, may result in altered drug metabolism capacity in NAFLD patients. On the other hand, genetic and association studies have indicated that a malfunction in drug metabolism can affect the prevalence and severity of NAFLD. This review focuses on the complex interplay between NAFLD pathogenesis and drug metabolism. A better understanding of these relationships is a prerequisite for developing improved drug dosing algorithms for the pharmacotherapy of patients with different stages of NAFLD. Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in Western countries with a wide disease spectrum. It ranges from the hepatic accumulation of lipids known as steatosis, non-alcoholic steatohepatitis (NASH) wherein steatosis is accompanied by inflammation and can further progress to cirrhosis and hepatocellular carcinoma. NAFLD is the hepatic manifestation of the metabolic syndrome as it is frequently associated with obesity, insulin resistance, hyperglycemia, hypertension, and dyslipidemia , and gene expression changes in cell development, morphology, movement, death, and antigen presentation pathways  are at a higher risk of developing NAFLD, with a prevalence of 85–98% for NAFLD and >25% for NASH  and ATP-binding cassette (ABC)-transporters are responsible for the hepatocellular uptake and excretion of xenobiotics. The expression of phase I and II DMEs and transporters is regulated by a battery of nuclear receptors in a coordinated manner . DMEs are classified as phase I, mainly cytochrome P450s (CYPs) that catalyze hydroxylation reactions, or phase II enzymes that are involved in conjugation reactions. Human phase I DME subfamilies CYP3A and CYP2C account for 50% of all hepatic CYPs and metabolize a large number of diverse drugs, e.g., lovastatin, tamoxifen, and R-warfarin. Phase II DMEs include UDP-glucuronosyltransferases (UGTs), sulfotransferases (SULTs), Non-alcoholic fatty liver disease patients show differences in drug metabolism and its regulatory components, as summarized in Tables Several drug metabolizing CYPs are downregulated in genetically modified  and diet-induced  NAFLD animal models and in patients with characteristics of the metabolic syndrome  null mice. POR is an essential redox partner of the microsomal CYPs. The liver conditional Por knockout mice display hepatomegaly, hepatic steatosis, and a reduced capacity of drug metabolism  index  is a ligand-activated nuclear receptor that upon activation forms a heterodimer with retinoid X receptor (RXR) and regulates the expression of a wide range of DMEs  is also a key regulator of xenobiotic and endobiotic metabolism , has been identified in the sequence flanking the start codon of FXR in European, African, Chinese, and Hispanic-American populations. It associates with decreased FXR expression and functionality, which may contribute to the pathogenesis of metabolic disorders  and Liver X receptor (LXR) are not major regulators of xenobiotic metabolism, but they play an important role in the metabolism of cholesterol and bile acids , carnitine palmitoyl transferase I (Cpt-I), mitochondrial β-oxidation enzymes, and hepatic lipases in addition to downregulating apoCIII and decreasing hepatic triglyceride secretion  are transcription factors that are activated by endogenous ligands, such as fatty acids, and synthetic ligands, such as the hypolipidaemic fibrates and the insulin-sensitizing thiozolidinediones. Like PXR and CAR, they form heterodimers with RXR and transactivate numerous target genes with vital roles in metabolism by binding to PPAR response elements , NAD(P)H:quinone oxidoreductase-1 (NQO1)] and activating their transcription . The expression of a canalicular biliary efflux transporter, multidrug resistance protein 2 (MRP2/ABCC2) and sinusoidal transporters, MRP3 and MRP4 is also regulated by NRF2 in mouse liver and HepG2 cells in response to oxidative stress and xenobiotics, thus providing further evidence that phase II enzymes and efflux transporters are regulated simultaneously  is a transcription factor that responds to oxidative/electrophilic stimuli by releasing from its repressor Kelch-like ECH associating protein 1 (Keap1) in the cytosol, translocating to the nucleus, binding to antioxidant response elements (AREs) upstream of numerous phase II DME genes, and genes involved in redox balance and oxidative stress response [e.g., heme oxygenase-1  results in decreased expression and activity of CYP3A4, with carriers of the T allele requiring significantly lower doses of statins  and 7% (CYP2C9*3). Both polymorphic alleles were associated with increased response to anti-diabetic sulfonylurea drugs and an increased glycemic response in T2D patients  and PPARγ coactivator-1 alpha (PGC-1α), which is also involved in energy homeostasis  on MCD diet display elevated Cyp2e1 expression that correlates with increased lipid peroxidation and oxidative damage, thus highlighting the role of CYP2E1 in the progression to NASH in response to increased hepatic free fatty acids -hydroxylase, catalyzes the oxidation of many low molecular weight molecules, including ethanol and acetone, a product of fatty acid oxidation. An important catalytic feature of CYP2E1 is the generation of ROS such as superoxide anion radical and hydrogen peroxide as a result of uncoupling of oxygen consumption with NADPH oxidation and as a by-product of lipid peroxidation  are present as different isoforms Alpha, Mu, and Pi and conjugate electrophilic compounds with reduced GSH  provides the cysteine moiety for the generation of GSH. Several rodent studies have indicated a decrease in SAM on a high-fat diet -null mice on a high-fat diet , organic anion transporting polypeptide 1a1 (OATP1a1), 1a4, 1b2, 2b1, OAT2, and OAT3. The expression of these transporters is significantly altered in the transition from steatosis to NASH rather than between control and steatotic samples (Fisher et al., Solute carrier transporters are uptake transporters that transport molecules from the blood into the hepatocyte. Studies in rat and human samples have indicated a coordinated downregulation of uptake transporter genes in NASH, such as the sodium/bile acid transporter (via the bile. ABC-transporters are the most extensively studied and are altered in steatotic and NASH livers (Buechler and Weiss, rs17222723 and rs8187710 variants in ABCC2 significantly associate with NAFLD and clinical and histological parameters (Sookoian et al., Transporters on the hepatocyte canalicular membranes are involved in the secretion of several endobiotics and xenobiotics The high prevalence of NAFLD is concerning in terms of general population health and also drug treatment regimens. A recent study in mice has identified that the feed-forward cycle of continuous exposure to high-fat diet over two generations leads to a significantly higher degree of obesity, NAFLD, insulin and leptin resistance, and epigenetic modifications resulting in increased lipogenesis and ER stress in future generations (Li et al., With the widespread prevalence of NAFLD, the proportion of patients with steatotic livers undergoing drug therapies for various disorders has also increased. The variability of drug treatment responses in these patients highlights the need for personalized therapeutic regimens. As detailed in this review, several components of the drug metabolism pathway are significantly affected in the presence of NAFLD. Similarly, genetic variations in DMEs and nuclear receptors associate with NAFLD with either positive or negative prognosis. Hence, inter-dependent interactions and common confounding factors exist between the pathogenesis of NAFLD and altered drug metabolism. As a majority of the DMEs are also involved in the metabolism of steroids and other lipids, polymorphisms in DMEs resulting in non-functional proteins may further aggravate the prognosis of NAFLD. The utility of identified genetic associations to determine NAFLD disease susceptibility, improve drug sensitivity or prevent adverse drug reactions holds great potential. Further efforts to characterize DMEs and identify risk factors for adverse drug reactions or treatment efficacies in NAFLD populations may lead to the utilization of innovative interdisciplinary strategies to provide a better insight into the pharmacokinetic profile of drugs and their efficacy. Although the implementation of these findings in the clinic is still a long-term goal with hurdles to pass, novel technologies and increasing interest in this field continues to increase our understanding of NAFLD and its interactions with drug metabolism.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Our understanding of global threats and local-scale disturbances on reefs is growing, but their relative contribution to reef resilience and vulnerability in the future is unclear. Here, we analyse quantitatively how different combinations of CO2 and fishing pressure on herbivores will affect the ecological resilience of a simplified benthic reef community, as defined by its capacity to maintain and recover to coral-dominated states. We use a dynamic community model integrated with the growth and mortality responses for branching corals (Acropora) and fleshy macroalgae (Lobophora). We operationalize the resilience framework by parameterizing the response function for coral growth  by ocean acidification and warming, coral bleaching and mortality by warming, macroalgal mortality by herbivore grazing and macroalgal growth via nutrient loading. The model was run for changes in sea surface temperature and water chemistry predicted by the rise in atmospheric CO2 projected from the IPCC's fossil-fuel intensive A1FI scenario during this century. Results demonstrated that severe acidification and warming alone can lower reef resilience  even under high grazing intensity and low nutrients. Further, the threshold at which herbivore overfishing (reduced grazing) leads to a coral–algal phase shift was lowered by acidification and warming. These analyses support two important conclusions: Firstly, reefs already subjected to herbivore overfishing and nutrification are likely to be more vulnerable to increasing CO2. Secondly, under CO2 regimes above 450–500 ppm, management of local-scale disturbances will become critical to keeping reefs within an Acropora-rich domain.Ocean warming and acidification from increasing levels of atmospheric CO We use established relationships between ocean chemistry and coral growth rates and between ocean warming, coral bleaching and mortality, as input functions into the resilience model for the benthic model reef community. We then run a series of simulations to address a key question: how will the resilience of this system vary under different ocean acidification, warming, and algal grazing scenarios? To answer this question we focus on determining the threshold levels of CO2 and grazing for when the community shifts from a coral-dominated regime  to a regime of alternate coral-algal states  and further to an algal-dominated regime .In the context of reef resilience, ocean acidification, reduced grazing and nutrification can be viewed as press-type processes that define axes of changing environmental conditions, whereas warming increases the frequency of acute (stochastic) perturbations , potentially shifting the reef community to low-coral states with increasing frequency and intensity. Here, we develop a novel mechanistic resilience model and analyse how increasing atmospheric CO2 and the local-scale disturbances overfishing and nutrification operate mechanistically in defining coral resilience. Our analytical approach builds on a parsimonious model for the dynamics of corals and macroalgae, using growth and mortality as the vital rates. Our approach does not provide absolute measures of coral and macroalgal abundance with high confidence, but allows an analytical comparison of the relative roles of environmental and ecological processes as drivers of resilience patterns – a comparison that would otherwise be intractable using more complicated community models , fleshy macroalgae (M) and ‘free’ space . While this simple system does not resolve the dynamics of complex reef communities, it allows questions to be addressed regarding the effect of complex environmental scenarios on reef resilience. The system is driven by three key processes: (i) mortality of corals and macroalgae, (ii) the rate at which corals and macroalgae grow and recruit into areas of free space, and (iii) the probability of competitive success by macroalgae over corals  ocean acidification primarily affects coral calcification, (2) warming mainly affects coral mortality via bleaching, (3) grazing affects algal mortality only and (4) nutrients affect the growth rate of algae only. We acknowledge that each process may have multiple secondary effects and we have outlined these with dashed lines  rates at which macroalgae and corals grow and recruit into areas of free space, respectively  to experimental growth data for A. intermedia on Heron Reef . We used the relative increase in buoyant weight of groups of A. intermedia branches (N=20 per treatment level) exposed for 8 weeks to three or four CO2 dosing regimes (affecting Ωarag) and two temperatures in a flow-through aquarium system  thus α and β are regression parameters. The fit of the model, which explained 83% of the variation in T and Ωarag is shown in Because the sum of corals, macroalgae and free space equal unity, free space (turfs) can be described as uveniles , our modcal seas  and datad T e.g. , we usedrC-base, multiplied by the ratio of projected net rates of calcification, Gnet, (forced by projected Ωarag and T) relative to the calcification rate at the study baseline (380 ppm), Gnet base  as a function of ocean acidification and warming, we used the baseline transition probability that corals can colonize free space, net base . Thus,  background mortality in the absence of bleaching, dC-base, (2) the amount of coral bleaching due to thermal stress, B , projected to increase in frequency and severity  or macroalgae (Lobophora) will colonize a free space (turfs) opened by a disturbance  that included the Austral summer for the southern Coral Sea  as CO2-forced input variables into coral growth and mortality models under the fossil-fuel-intensive A1FI scenario by the IPCC (2 and associated Ωarag and T were generated using the University of Victoria Earth system model (UVic), which simulates the global climate and carbon cycle  for six sites in the region  were increased in 20 increments from 380 to 1000 ppm within each of three herbivore grazing rates on macroalgae  to analyse explicitly the effects and potential interactions of ocean acidification, warming and grazing on the resilience of the simplified coral reef community used here. Rather than identifying the location of community equilibria, which are unrealistic by assuming the absence of acute (stochastic) disturbances, we estimated the frequency distribution of community states  during repeated (1000 model runs) 50-year community projections within each of the 20 CO2 bins and for each grazing scenario. The frequency and severity of stochastic disturbances such as thermal anomalies and cyclones were included in projections by modelling the variation in bleaching-induced mortality, dCB, and background coral mortality, dC-base, as Poisson distributions with means of 30% and 10%, respectively, and standard deviations of 10%  macroalgal growth, γ, from varying nutrient loading, (3) coral mortality from bleaching, dCBl, and (4) competitive strength of macroalgae over corals, a, we ran a series of sensitivity analyses in which coral abundance projections were generated using parameter values from either end of the range used for each core parameter , and for high (60%) and low (30%) herbivore grazing rate. All model programming was developed using matlab 7.8 .To examine the robustness of the projected resilience patterns to changes in the values of the core vital rate parameters, (1) coral growth, arameter . For traAcropora/Lobophora/turf system indicate that ocean acidification and warming are critical drivers of change in coral resilience via impacts on coral growth rates and survivorship. For this model community system, reefs characterized by Acropora assemblages with low background mortality (Lobophora), mean coral abundance was projected to fall by more than 50% by the highest CO2 level  coral-dominance , (2) alternate coral–algal states  and (3) algal-dominance . Reducing grazing rates from 60% to 40% (e.g. simulating increased fishing of herbivores) lead to a decline in coral resilience in response to increasing CO2  and low grazing (30%) led to a shift characterized by near complete coral loss and an increase in macroalgal cover of up to 50%  in the decline of coral resilience . Specifically, increasing COsing CO2 . SpecifiO2 level  and 5b. level , indicated that coral resilience patterns in 2 and grazing. Detailed analyses, however, indicated that coral abundance projections had different sensitivities to different parameters, depending on CO2 and grazing scenario. Interestingly, whereas coral abundance projections were sensitive to coral growth for all combinations of CO2 and grazing, they were only sensitive to the other three parameters for some combinations of CO2 and grazing. Specifically, perturbing rC-base by 30% to either side of its mean value shifted the mode of the coral abundance peaks by 5–20% along the coral cover axis for most CO2 and grazing scenarios. As coral growth rate was assumed to be driven by water carbon chemistry and temperature regime only, this sensitivity partly represented uncertainty in the effects of acidification and warming. However, sensitivity to coral growth variation was highest at high grazing and high CO2 where corals were predicted to dominate, although at low abundance  was predicted to promote a shift from coral dominance to a regime of alternate coral and algal states  incurred a drop in coral abundance to below 20% cover. Under low grazing and high CO2, variation in bleaching mortality determined the extent to which coral abundance projections formed alternate coral-algal states or led to coral loss and algal dominance  and fleshy macroalgae (Lobophora), that the effects of ocean acidification and warming on coral growth and mortality will have important impacts on coral reef resilience under increasing CO2. Specifically, by reducing coral growth (due to acidification) and survivorship (due to warming), increasing CO2 will lower the threshold value at which local and regional processes like herbivore overfishing and nutrification drive the study community from predominantly coral-dominated to predominantly algal-dominated states. Therefore, warming, acidification, overfishing and nutrification all drive the dynamics of the system in the same direction, suggesting that reduced coral resilience in a high-CO2 world is likely to be a consequence of both global threats and local-scale disturbances.Overfishing and/or mass mortality of herbivores  are consAcropora abundance to less than half the current abundances despite high rates of grazing and low levels of nutrification. This indicates that management efforts to maintain healthy herbivore populations and high water quality will become increasingly critical as atmospheric CO2 levels rise – and that such efforts could be futile in the longer-term if efforts to curb emissions growth are unsuccessful. Thus, even coral reef systems with effective management systems in place such as the Great Barrier Reef (2 scenario (e.g. below 540 ppm) local management effective in maintaining or restoring healthy herbivore populations (high grazing) and low nutrients can increase the chances that reefs will remain coral-dominated. Based on the likely shift from coral dominance to algal dominance under the high CO2 and low grazing scenario in 2 and high grazing, the combination of high CO2 and low grazing (herbivore loss) leads to severe coral loss even under the highly conservative assumptions made here.These findings have far-reaching implications for the health of coral reefs in the future for at least two reasons. Firstly, under a fossil-fuel intensive carbon emission path (the A1FI scenario by the IPCC) – the current global trajectory – acidification effects on coral calcification and increased coral mortality from bleaching may potentially reduce ier Reef  are pred2 will exacerbate effects of overfishing and nutrification on reef ecosystems. However, by formally accounting for interactions between CO2 effects and local-scale processes on reef state probability under both pulse and press disturbance regimes, our model analyses provide novel quantitative estimates of resilience patterns and allow assessments of the relative contribution from environmental and ecological processes in maintaining resilience. Although our projections of Acropora abundance as a function of CO2 level do not constitute accurate predictions, they reflect the relative impact of various disturbances on a simplified reef community.The results of this study generally support the conclusions of recent reviews (e.g. rC) and assume that the growth rate of macroalgae (parameter γ) of the type used here (fleshy brown Lobophora sp.) will not be affected by acidification or warming. Results of recent experiments, however, indicate that the rate of linear extension of Lobophora is 20–40% enhanced under intermediate CO2 levels  will remain unchanged under varying CO2. Again, recent experiments indicate that ocean acidification strongly shifts the competitive coral–algal interaction in favour of the macroalgae , suggesting that a will increase with ocean acidification. Thirdly, our model assumes that rates of coral calcification are unaffected by bleaching, which is likely to overestimate coral growth rates under high CO2 levels where corals are likely to bleach  and local-scale disturbances (overfishing and nutrification) will both contribute to reducing the resilience of corals this century. Also, our findings that the abundance of corals under high CO2 is sensitive to variation in coral growth and mortality parameters as well as to variation in algal growth and competition parameters indicate that coral resilience patterns are driven by both CO2  and nutrification . The increased sensitivity of the model projections for corals to variation in algal growth under low grazing (herbivore overfishing) indicates that nutrification effects on coral resilience becomes particularly important in the proximity of threshold points for grazers control of algal biomass (2 and low grazing suggests that algal-dominated and coral-depauperate systems are persistent. While the difficulty in reversing shifts from coral- to algal-dominance has been long known (2 world.Given the conservative assumptions listed here, the results of our model projections strongly suggest that the increase in atmospheric CO2. Firstly, a failure to rapidly stabilize and reduce the concentration of CO2 in the Earth's atmosphere is likely to lead to significant loss of key framework builders such as Acropora, irrespective of the effectiveness of local management. Secondly, local reef management efforts to maintain high herbivore grazing and low nutrients have the potential to play a critical role in maintaining coral resilience while CO2 concentrations are stabilized.In conclusion, our quantitative analyses of resilience patterns for a simplified model system bring into focus two core principles regarding the future of coral reefs under increasing CO


Protein aggregation, mitochondrial deficits, oxidative and autophagic stress are prominent pathologies in neurodegenerative diseases. Our laboratory has a long term interest in the regulation of autophagy-lysosomal activities in neurodegeneration. We previously found that deficiency in the lysosomal cathepsin D in a mouse knockout model led to accumulation of the key protein in Parkinson’s disease, α-synuclein. Overexpression of cathepsin D in human neuroblastoma cells as well as worms reduced α-synuclein aggregation and toxicity. Recent work found that enhanced expression of lysosomal cathepsin D reduced both full-length and fragmented huntingtin in transfected HEK cells, and protected against mutant huntingtin toxicity in primary neurons. Furthermore, as mitochondria are the major generators and targets of reactive species, and oxidative stress has been found in a wide range of neurodegenerative diseases, investigating the source and nature of oxidative stress in cathepsin deficient brains are of significant interest. It has been previously shown that α-synuclein modification by S-nitrosylation enhances its aggregation propensity. Furthermore, microglial produced nitrous oxide (NO) from an increase in inducible nitrous oxide synthase (iNOS) underlies some of the brain pathologies in CD knockout mice. Given that other lysosomal storage conditions have demonstrated mitochondrial involvement and that oxidative and nitrative changes have been associated with CD deficiency, we studied the effects of CD deficiency on mitochondrial morphology and function with the intent to understand how this lysosomal enzyme play a role in maintaining cellular bioenergetics and management of cellular oxidative states. We have found that although there are no changes in total mitochondrial biomass due to CD deficiency, there are enlarged mitochondria, and deficits in functional parameters of the electron transport chain. The changes demonstrated in this study contribute to the understanding of how mitochondrial/lysosomal interplay affects the pathogenesis of neurodegenerative diseases.


In humans and other multicellular organisms that have an extended lifespan, the leading causes of death are atherosclerotic cardiovascular disease and cancer. Experimental and clinical evidence indicates that these age-related disorders are linked through dysregulation of telomere homeostasis. Telomeres are DNA protein structures located at the terminal end of chromosomes and shorten with each cycle of cell replication, thereby reflecting the biological age of an organism. Critically shortened telomeres provoke cellular senescence and apoptosis, impairing the function and viability of a cell. The endothelial cells within atherosclerotic plaques have been shown to display features of cellular senescence. Studies have consistently demonstrated an association between shortened telomere length and coronary artery disease (CAD).Several of the CAD risk factors and particularly type 2 diabetes are linked to telomere shortening and cellular senescence. Our interest in telomere biology was prompted by the high incidence of premature CAD and diabetes in a subset of our population, and the hypothesis that these conditions are premature-ageing syndromes. The assessment of telomere length may serve as a better predictor of cardiovascular risk and mortality than currently available risk markers, and anti-senescence therapy targeting the telomere complex is emerging as a new strategy in the treatment of atherosclerosis. We review the evidence linking telomere biology to atherosclerosis and discuss methods to preserve telomere length. Atherosclerosis is an age-related disorder.The histology of atherosclerotic plaques has been comprehensively studied and has demonstrated that endothelial and vascular smooth muscle cells in atherosclerotic lesions display changes of senescence.Telomeres are DNA protein structures located at the extreme ends of the chromosomes.They cap and protect the ends of chromosomes. Whereas the DNA molecule carries the genetic code and is about 100 million base pairs long, the telomeric ends are non-coding and are between 5 000 and 15 000 base pairs long: 15 000 at the time of human conception and around 5 000 at the time of death.8During DNA replication, the very end sequences of the telomere are not fully copied due to the inability of DNA polymerase to completely replicate the chromosome to its very end. This is termed the end-replication problem. As a result, between 50 and 200 nucleotides are lost with each cycle of cell replication, leading to progressive telomere shortening.Cellular senescence and the apoptotic cascade are mediated by cell cycle checkpoint pathways, regulated mainly by p53/p21, which are best recognised as tumour suppressor proteins.11However, a limited number of cells (about one in 10 million) are able to reactivate the enzyme telomerase. In the presence of telomerase, cells are able to replicate and in this way telomere integrity is maintained. Telomerase activity is lacking in somatic cells but is preserved in reproductive and stem cells. High telomerase activity has also been detected in about 90% of human cancer samples. The high telomerase activity is thought to be responsible for the indefinite cell proliferation and cellular immortalisation seen with cancer.Studies have shown that telomere length is not only determined by cell replication and lifespan, but is also influenced by heredity and exposure to environmental risk factors. The healthy offspring of parents with coronary artery disease have shorter telomeres than the offspring of normal subjects.Diabetic patients, more than any other subset, show the greatest difference in telomere length compared to non-diabetics.26The study of telomeres may therefore provide in a single marker, the combined influence of genetics, environmental risk and ageing in predicting risk and identifying susceptible individuals prone to developing coronary artery disease. This is especially relevant in our community, which has a high incidence of both premature coronary artery disease and type 2 diabetes.30Telomeres have a dynamic structure that is thought to switch between a closed, protected state and an open, extendable state, which allows the DNA terminus to undergo replication. The protected state is necessary for safeguarding the integrity of genomic material, whereas the extendable state allows the enzyme telomerase to extend short telomeres Figs , 2.31 Te• The DNA component: this consists of tandem repeats of the hexanucleotide 5′-TTAGGG-3′  and has a high guanine content. The bulk of telomeric DNA is arranged in the double-stranded configuration, which then ends in a single-stranded extension. The single-stranded overhang folds back to form a terminal loop, which prevents the end of the telomere from being recognised as a damaged, broken end. Telomere shortening is thought to destabilise this loop.31• Shelterin proteins: these proteins bind and protect the loop structure and are termed shelterin because they shelter the chromosome end.32 An inability to form the terminal loop will leave the chromosome ends uncapped, resembling a DNA break and provoking DNA repair mechanisms. The shelterin complex consists of six proteins, which have specific functions in telomere replication and end protection.The six proteins are: TRF1 and TRF2: telomere repeat-binding factors 1 and 2, which are the two major proteins; POT1: protection of telomeres 1; TPP1: tripeptidyl peptidase 1; TIN2: TRF1-interacting protein 2; and RAP1: repressor activator protein 1. Whereas the shelterin proteins are a constant fixture at the telomere end, other accessory proteins are intermittently recruited to the telomere. These proteins include the tankyrases tank 1 and 2, Ku 70/86 and poly-ADP ribose polymerase-1 (PARP-1), which influence the control of telomere length and repress the DNA damage response.34• The CST complex: an additional telomere-associated complex, known as the CST, has recently been identified. It binds single-stranded DNA and appears important for both telomere protection and replication.31• Telomerase: in order for cellular repair to take place as well as for species survival, stem cells and reproductive cells need to be able to proliferate without the penalty of progressive telomere shortening.Telomere length in proliferating cells is influenced by the following factors.• Factors that shorten telomeres:- telomere attrition during cell division- DNA damage due to oxidative stress caused by environmental risk factors- specific exonucleases involved in the degradation of RNA primers used for DNA replication- deficiency of Rad 54, which is involved in DNA repair- histones: methylation of histones H3 and H4 diminishes telomerase activity.36• Factors that maintain telomere length:- Telomerase: in addition to the level of telomerase within a cell, telomere length is also dependent on the delivery of telomerase to the telomere by Cajal bodies, telomerase access to the DNA terminus and the presence of molecules that stimulate or inhibit telomerase activity.31- A recombination process known as alternative lengthening of telomeres or ALT (10% of cancers maintain their telomere length by ALT).37The two major mechanisms responsible for telomere shortening are the end-replication problem, and more importantly, the oxidative DNA damage induced by environmental risk factors. Telomere shortening due to the end-replication problem is relatively small and constant in each cell, irrespective of telomere length, whereas telomere shortening induced by oxidative stress is proportional to telomere length, as longer telomeres are larger targets for free radicals.40Variability in telomere length is also noted at birth and is influenced by heredity, race and gender. Telomere length has been shown to be shorter in healthy offspring of patients with coronary artery disease (CAD).452-), a two-electron reduction forms hydrogen peroxide (H2O2), and a three-electron reduction forms the hydroxyl radical (OH). Many other ROS species can be derived from superoxide and hydrogen peroxide.Oxidative stress is the unifying pathophysiological mechanism responsible for ageing and age-related disorders.These ROS initiate processes involved in atherogenesis through several enzyme systems including xanthine oxidase, NADPH (nicotinamide adenine dinucleotide phosphate) oxidases and nitric oxide synthase.• Decreased availability of nitric oxide (NO), which results in defective endothelial vasodilation. Nitric oxide is an antiatherosclerotic agent that protects vascular cells from apoptosis.53• Inflammation: ROS increase the production of pro-inflammatory cytokines such as tumour necrosis factor alpha (TNF-α), which in turn can also increase the production of ROS. TNF-α activates two transcription factors: nuclear factor kappa-β (NF-κβ) and activator protein-1 (AP-1), which increase the expression of pro-inflammatory genes. Cytokines stimulate the synthesis of acute-phase reactants such as C-reactive protein (CRP) by the liver. ROS also increase the expression of cellular adhesion molecules on the endothelial cell surface. These molecules, intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), enhance monocyte adhesion to endothelial cells and lead to the formation of atherosclerotic plaques.58In vitro studies have shown that LDL cholesterol (LDL-C) is not atherogenic in itself but it is the oxidative modification of LDL-C that plays a critical role in the pathogenesis of atherosclerosis.62• Modification of lipoproteins and lipids: ROS contribute to the formation of lipid peroxides, which bind to proteins to form advanced lipoxidation end products (ALEs).This pathway has an important regulatory role in cellular proliferation and survival. Of the three known isoforms of Akt, Akt 1 is most relevant in regulating cardiovascular cell growth and survival and Akt 2, which is highly expressed in muscle and adipocytes, contributes to regulation of glucose homeostasis. These isoforms are activated by growth factors, extra-cellular stimuli such as pro-atherogenic factors and by oncogenic mutations in upstream regulatory proteins. Akt mediates downstream signalling pathways through phosphorylation of a host of substrates. Thus far, more than a hundred substrates for Akt have been identified, indicating that it has widespread biological effects. Dysregulation of Akt is associated with cardiovascular disease, diabetes, cancer and neurological disorders.et al. have demonstrated that pro-atherogenic factors such as ox-LDL, TNF-α and hydrogen peroxide promoted endothelial cell senescence by inactivation of the PI3/Akt pathway. Akt was shown to maintain telomerase activity by phosphorylation of its TERT subunit, and inactivating Akt reduced telomerase activity, leading to accelerated endothelial cell senescence.63Our current understanding of its role in cardiovascular disease is incomplete and studies explaining its effects describe conflicting mechanisms. Breitschopf et al. demonstrated that activation of Akt promotes senescence and arrests cell growth via the p53/p21-dependent pathway and that inhibition of Akt extends the lifespan of primary cultured human endothelial cells. Akt achieved growth arrest by phosphorylating and inhibiting a forkhead transcription factor (FOXO 3a), which influences p53 activity by regulating levels of ROS.et al. confirmed the latter mechanism by demonstrating that endothelial progenitor cells cultured in the presence of ox-LDL in a diabetic milieu underwent senescence and growth arrest by activation of the Akt pathway via accumulation of p53/p21.65On the other hand, Miyauchi et al. commented that the divergent observations may be explained by the different cell types used in studies. They used primary human endothelial cells, whereas most other studies examined immortal cells in which the normal cell cycle machinery may have been impaired. In addition, Akt may promote cell proliferation or senescence depending on other factors such as the duration and extent of its activation. It has been noted that activation of Akt in itself is insufficient to cause cancer unless combined with other oncogenic stimuli.Miyauchi There is currently much interest in the development of Akt inhibitors for the treatment of cancer and it remains to be seen what effects such therapy would have on the cardiovascular system. In addition to Akt signalling, mitogenic stimuli may activate Ras signalling, which has also been shown to participate in the divergent processes of both cell proliferation and senescence.66Exposure of DNA to oxidative stress produces higher levels of stress biomarkers in telomere sequences than in non-telomere sequences. 8-oxodG  is a sensitive biomarker for oxidative stress on DNA. Progressive increases in 8-oxodG have been shown to correlate with decreasing telomere length. The high guanine (-GGG) content of telomeres makes them particularly sensitive to damage by oxidative stress.67This site specificity for guanine is due to several reasons. Firstly, guanine is the most easily oxidised DNA base as its oxidation potential is lower than that of the other three bases (adenine < cytosine < thymine). A second factor is the distribution of electrons on the DNA base. The highest occupied molecular orbital that accommodates electrons with the greatest energy determines the reactivity of DNA bases. Many of these electrons are located on the 5′-G of the GG sequence and therefore this guanine is more likely to be oxidised.A third reason is that the ROS have different redox potentials, which may determine site specificity. For example, the free hydroxyl radicals cause DNA damage without a marked site specificity, whereas the benzoyloxyl radicals specifically cause damage to the 5′-G in GG sequence.Chronic systemic inflammation is responsible for an increase in peripheral white blood cell turnover, which in turn leads to an exaggerated telomere attrition rate.57et al. demonstrated that telomere attrition in circulating blood leucocytes reflects similar changes in the vasculature and is an acceptable surrogate for vascular ageing in population studies.72DNA sampling for telomere length quantification is generally sourced from circulating white blood cells rather than human vascular tissue. It has been suggested that white blood cell telomere attrition is a consequence of systemic inflammation rather than being indicative of vascular endothelial cell ageing. The study by Wilson The atherosclerotic process is characterised by endothelial cell dysfunction. Repair of the endothelium is dependent on the presence of endothelial progenitor cells, which migrate to sites of vascular injury to initiate repair. Endothelial progenitor cells are produced by haematopoietic stem cells, which, due to their higher telomerase activity, have a greater proliferative capacity. Exhaustion of the progenitor cell or stem cell pool is an important factor in endothelial cell dysfunction. Telomere length in haematopoietic stem cells (HSC) is a reflection of progenitor cell reserves, and shortened telomere length in these cells is indicative of diminished reparative capacity.42The onset of atherosclerotic disease is therefore dependent on the balance between injury and repair of the endothelium – injury from oxidative stress and inflammation, and repair, which depends on haematopoietic stem cell reserves, as reflected by HSC telomere length.41et al., reporting on the Flemish study on environment, genes and health outcomes, found shorter telomeres in smokers compared to non-smokers.et al. showed that women who had never smoked had longer telomeres than former smokers, and both had longer telomeres than current smokers .Cigarette smoking is associated with increased oxidative stress.et al. who studied a cohort of male smokers with and without chronic obstructive pulmonary disease  in whom telomere shortening correlated with cumulative exposure to tobacco smoking.20They also demonstrated a dose-dependent relationship between smoking and telomere shortening. Each pack-year smoked was equivalent to the loss of an additional five base pairs of telomere length, or 18% of the average annual loss in telomere length, compared to the rate in the overall cohort.et al. postulated that arterial pulse pressure may correlate with biological age. Among 49 twin pairs (mean age 37 years) in the Danish Twin Register, they showed a significant inverse correlation between pulse pressure and telomere length, i.e. wider pulse pressure was associated with shorter telomere length.74Since systolic blood pressure rises with age, and diastolic blood pressure plateaus, Jeanclos n = 171) compared to their normotensive peers (n = 156) but the shorter telomere length was largely due to insulin resistance.et al. examined the relationship between telomere length and carotid artery atherosclerosis in 163 treated hypertensive males and found that telomere length was shorter in hypertensive men with carotid plaques compared to hypertensive men without plaques.75The Framingham Heart Study found shorter telomere lengths in hypertensive males  and telomere attrition rate.22et al. of 1 122 healthy adult female twins , it was found that the telomeres of obese twins were 240 base pairs shorter than those of the lean sibling. The difference in telomere length between the lean and the obese corresponded to 8.8 years of ageing.per se.In the study by Valdes et al. confirmed the negative association between BMI and telomere length but in their study, this finding applied only to female participants.81Obesity is associated with high serum concentrations of leptin, which is linked to NF-κB activation, a mediating factor in the production of ROS and inflammatory cytokines.Insulin resistance is pro-atherogenic and increases the risk of CAD even without the presence of hyperglycaemia.In early atherosclerosis, insulin resistance causes decreased nitric oxide production and an increase in VCAM-1, which are responsible for impaired vasodilation and inflammation. In advanced plaques, insulin resistance triggers apoptosis of cells via the Akt pathway.In the setting of type 2 diabetes, insulin resistance and hyperglycaemia have additive effects that accelerate the process of atherosclerosis. Hyperglycaemia is associated with the activation of several molecular pathways that include the production of advanced glycation end products (AGEs),It is well established that hyperglycaemia, even in the pre-diabetic state, induces oxidative stressIn recent years several cross-sectional clinical studies have been published that demonstrate an association between shorter telomere length and type 2 diabetes (T2D).100et al. showed that CAD patients with the metabolic syndrome had shorter telomeres than CAD patients without the metabolic syndrome.et al. found that among diabetic patients, those with atherosclerotic plaques had shorter telomeres.et al. demonstrated that diabetic patients with myocardial infarction had shorter telomeres than diabetic subjects without myocardial infarction,et al. showed that among diabetic subjects, those with CAD had significantly shorter telomeres.26Satoh Based on these observations, it has been postulated that critically shortened telomeres, due to a combination of inherited short telomeres and oxidative stress-induced telomere attrition, caused by the common risk factors between diabetes and cardiovascular disease, indicates greater cellular ageing in vascular endothelial cells and pancreatic beta-cells, and may be a useful biomarker of tissue ageing and disease progression.100et al. have shown that endothelial cells with characteristic features of senescence are present in atherosclerotic regions of human coronary arteries. They demonstrated that inhibiting telomere function induced senescence in endothelial cells, whereas introducing telomerase suppressed senescence and extended the lifespan of these cells.3Minamino et al. have shown that the telomeres of coronary endothelial cells were shorter in patients with CAD compared to age-matched subjects without CAD and that in the CAD patients, telomere length was shorter in endothelial cells at atherosclerotic sites compared to non-atherosclerotic sites.102Ogami et al. also demonstrated that telomere attrition was higher in the intima of the distal abdominal aorta compared to the proximal abdominal aorta, again indicating that areas of the vasculature that undergo greater shear wall stress have higher cellular turnover rates and consequently shorter telomere length.Okuda et al. of 10 cases and 20 control subjects, it was observed that mean telomere length was significantly shorter in patients with severe triple-vessel CAD compared with matched subjects who had normal coronary angiograms.106Population studies have demonstrated a link between telomere length and CAD.A retrospective registry analysis of 383 patients  showed that patients with premature myocardial infarction had significantly shorter mean telomere lengths. In this study the difference in telomere length between cases and chronologically age-matched controls demonstrated a biological age gap in excess of 11 years. Compared with subjects in the highest quartile for telomere length, the risk of myocardial infarction was increased between 2.8- and 3.2-fold in subjects with shorter-than-average telomeres.108In a sub-study of the West of Scotland Primary Prevention Study (WOSCOPS) that compared telomere lengths at recruitment in 484 individuals who went on to develop coronary heart disease events with those from 1 058 age-matched controls who remained free of CAD, it was shown that subjects with shorter telomere length at the time of recruitment had a significantly higher risk of developing subsequent coronary heart disease.110et al. measured telomere length in 780 patients with stable angina in a prospective cohort study. During a mean follow up of 4.4 years, shorter telomere length was significantly associated with all-cause mortality, independent of age, clinical and echocardiographic variables.et al. using samples collected at baseline in the prospective Physician′s Health Study from a cohort of 14 916 initially healthy men, of whom 337 went on to develop myocardial infarction, demonstrated that participants with shorter telomere length at baseline had a significantly increased risk of incident myocardial infarction compared to age- and smoking-matched controls who remained free of vascular disease over a mean follow up of 3.85 years.112Farzaneh-Far Finally, in the prospective population-based Bruneck study, baseline telomere length was a significant risk predictor for subsequent myocardial infarction and stroke, independent of standard risk factors. Of note in this study was that telomere length was strongly associated with advanced pathology and acute vascular syndromes but not early atherosclerosis.7Telomerase has been shown to be activated by lifestyle choices that include a healthy diet, stress relief through meditation, chronic high-intensity aerobic physical exercise as well as by pharmacological agents.114et al. in a recent study have shown that maintaining high levels of aerobic fitness preserved telomere length.2max levels.Exercise has been associated with improved cardiovascular health and longevity. La Rocca The molecular mechanisms exploring the protective effects of exercise on the heart has been studied in experimental animals. Exercise has been shown to promote cell survival by increasing the activity of telomerase and the expression of TRF2. The up-regulation of telomerase was mediated via insulin-like growth factor 2 and endothelial nitric oxide synthase. Exercise was also shown to decrease levels of markers of cellular growth arrest and apoptosis, such as p16, cell cycle-checkpoint kinase 2 and p53.Ginko biloba and resveratrol have been shown to activate telomerase, the latter two via PI3k/Akt signalling pathways. The anti-oxidants N-acetylcysteine and α-tocopherol enhance telomerase activity.et al. demonstrated in a prospective study of patients with stable CAD, an inverse relationship between baseline blood levels of marine omega-3 fatty acids and the rate of telomere shortening over five years.118Molecules that enhance low residual telomerase activity or re-express silenced telomerase may help preserve telomere length. Natural products such as derivatives from the Chinese Astragalus plant, Aspirin, ACE inhibitors and particularly statin therapy have been shown to positively impact on the vascular endothelium via anti-senescence effects. Over and above its anti-thrombotic and anti-inflammatory effects, aspirin has been shown to decrease the formation of dimethylarginine, an endogenous inhibitor of nitric oxide synthase, thereby reducing oxidative stress and delaying endothelial cell senescence.120et al. have shown that statins enhance the migratory capacity of endothelial progenitor cells by up-regulation of TRF2, the telomere-binding protein that stabilises telomere structure at the t-loop.et al. who studied patients presenting with acute myocardial infarction, showed that prior statin therapy was independently associated with significantly longer telomere length in subjects below the age of 64 years.123Several studies have suggested that the survival benefit attributed to statin therapy may be linked to its effects on telomere biology. Spyridopoulos Our interest in telomere biology stems from the high incidence of both premature CAD and type 2 diabetes mellitus witnessed in the population that we serve. Patients with CAD who have diabetes have worse outcomes than those without diabetes. This, coupled with the fact that the initial presentation in a substantial majority of our young patients is with myocardial infarction, which carries a worse prognosis than stable CAD, further contributes to adverse long-term outcomes. Indications are that revascularisation procedures are not as efficacious in this population.The availability of quantitative polymerase chain reaction, which is a simpler, less labour-intensive and cheaper method requiring smaller quantities of DNA compared to the standard method of southern blot analysis, has made it feasible for us to determine telomere length in our patients.125The study of telomere dynamics may serve several functions. Firstly, measuring telomere length in the early years of life may indicate a genetic predisposition and help target susceptible individuals. Studies on the genetic contribution to premature CAD with genome-wide association scans have yielded little thus far, whereas an assessment of telomere length provides a more universal insight into the genetics of CAD.Secondly, telomere length is a measure of cumulative DNA damage from multiple environmental risk factors over an individual’s lifespan and is likely a better predictor of CAD than the currently available risk markers, which are single, point measurements in time.Thirdly, although the development and progression of atherosclerosis occurs over decades, the process is clinically silent until the manifestation of full-blown disease. The rate of telomere shortening is accelerated prior to the onset of clinical disease, so longitudinal assessments of telomere length may be of predictive value. Finally, novel therapies aimed at delaying cellular senescence by manipulation of the telomere/telomerase complex may be of benefit.


Uncoupling proteins (UCPs) belong to a family of mitochondrial carrier proteins that are present in the mitochondrial inner membrane. UCP1 was first identified followed by its two homologs, UCP2 and UCP3. The physiological functions of UCP include lowering mitochondrial membrane potential and dissipating metabolic energy as heat. However, UCP can be dysregulated and may contribute to the pathogenesis of metabolic disorders and obesity. Recent studies suggest that UCP also plays a role in neurodegenerative diseases and atherosclerosis. In addition, the widely expressed UCP, UCP2, has been shown to be upregulated in a number of aggressive human cancers. One mechanism of UCP2 upregulation in these cancers is due to oxidative stress, and elevated UCP2 in turn reduces oxidative stress, which provides a growth advantage for these cancers. Nevertheless, new studies suggest UCP2 may interact with oncogenes and tumor suppressor genes, providing a potential new mechanism of how UCP2 contributes to cancer development. In this review, the evidence supporting the role of UCPs in diseases other than diabetes and obesity, the reports on how UCP is regulated in cancer cells, and how UCP may regulate p53 will be discussed. However, not all of the energy available in the electrochemical gradient is coupled to ATP synthesis. Some of the energy is consumed by “proton leak” reactions, by which protons pumped into the inner membrane space flow back into the matrix through proton conductance pathways in the inner membrane that bypass the ATP synthase. As a result, energy derived from the metabolic oxidation reaction is dissipated as heat [In vertebrates, energy is mainly produced in the mitochondria via the Citric Acid Cycle coupled with oxidative phosphorylation. The mitochondrial respiration process generates a proton gradient across the mitochondrial inner membrane that establishes the electrochemical potential , among those, UCP1 is the first to be identified in brown adipose tissue , followe2.Mitochondria-generated ATP provides the major fuel for eukaryotic cells. As byproducts, free radicals can be generated within mitochondria during energy production, which could harm cells if not rapidly removed. Mild mitochondrial uncoupling can reduce the generation of free radicals and therefore, protect cells. Mitochondrial uncoupling has been amplified or reduced during the pathogenesis of a number of human diseases due to up- or down-regulation of UCP. The two most studied UCP-associated diseases are diabetes and obesity, and studies of UCP in these two diseases have been thoroughly reviewed.et al. detected UCP2 in young Asian patients, and the results showed that the expression levels of UCP2 were correlated with general pathologic severity [Non-alcoholic fatty liver disease is another form of metabolic disorder. Park severity .et al. reports that the upregulation of UCP2 protects the liver from excessive fat accumulation, but chronically depletes the liver of ATP, compromising the response of the liver to acute energy demands, which results in increased susceptibility to ischemia-reperfusion injury [Non-alcoholic steatohepatitis (NASH) has been identified in patients with non-alcoholic fatty liver disease with evidence of inflammation in liver biopsies. The mechanistic progression of non-alcoholic fatty liver disease to non-alcoholic steatohepatitis is not clear; however the involvement of underlying mitochondrial dysfunction orchestrates the progression due to its role in fatty acid oxidation, ROS generation and ATP synthesis. NASH has been characterized by the accumulation of fatty acids in hepatocytes and oxidative stress , decreasn injury .etc.Clinical evidence suggests that UCPs may be associated with diseases distinct from metabolic diseases. For instance, higher expression levels of UCP2 may mediate follicle development in polycystic ovary syndrome . Severalet al. reported the presence of UCP2 mRNA with varying intensities throughout the brain. Marked high intensities were found in the hypothalamus, the ventral septal region, the ventricular region and the cerebellum, suggesting a novel role of UCP in modulating neuroendocrine functions and autonomic responses of the brain [Richard he brain . In addihe brain .This reghe brain .10 (CoQ10), a cofactor of mitochondrial metabolism and UCP2 activity [10 was shown to reduce dopamine cell loss in both mouse [The mRNA expression of UCP2 in the brain suggested neuronal localization . In an aactivity , inducedactivity . When gith mouse  and primth mouse  and poteth mouse . As mentth mouse . These sth mouse .Not surprising, UCP also plays an important role in Alzheimer’s disease (AD). The expression levels of UCP2, 4, and 5 were significantly reduced in AD patients, which were accompanied by upregulation of nitric oxide synthases (NOS), and resulted in increased oxidative stress and impaired mitochondrial functions. Therefore, to increase the expression level of UCP may help in the treatment of AD .LDLR-/-). These mice were transplanted with bone marrow from either UCP2 deficient mice or wild-type mice, and fed with an atherogenic diet. The results showed that the atherosclerotic lesion size was markedly increased in the mice receiving UCP2 deficient bone marrow, which was accompanied by enhanced oxidative stress [The protective role of UCP2 against atherosclerosis was demonstrated using irradiated low-density lipoprotein receptor-deficient mice  in cancer was recently recognized and has attracted more attention. As a matter of fact, the impact of mitochondrial uncoupling on cellular physiology is not restricted to normal cells; it also plays an important role in the reprogramming of cancer cell metabolism .Early evidence showing that UCP might be important for tumor growth arises from rats which received an inoculation of the Yoshida AH-130 ascites hepatoma cells . The expet al. reported that UCP2 overexpression in malignancy was limited to the colon [In human studies, most of the results point to UCP2 potentially being associated with the development of colon and breast cancer. Horimoto he colon , in whicet al. further demonstrated that the levels of both UCP2 mRNA and protein were higher in colon cancer tissue samples than in its adjacent tissue samples [Kuai  samples , and theHowever, the first colon tumorigenesis study using UCP2 null mice showed surprising results . The UCPet al. reported a significant association of UCP2 with tumor grade in primary breast cancer [et al., which demonstrated that UCP2 expression was correlated significantly with histological grade and mitotic count in invasive ductal carcinoma of the breast analyzed from human tissue microarrays [Later studies suggest that UCP2 is also involved in the pathogenesis of other cancers. Sayeed t cancer . The resroarrays .We used a tumor promotion model to study the role of mitochondrial uncoupling in tumor promotion . Our stuAnother important role of UCPs in cancer is their contributions to chemoresistance ,32. UCPs4.UCP gene regulation is not well understood. UCP can be generally regulated by oxidative stress. When oxidative stress occurs, UCP2 and UCP3 uncoupling activities can be stimulated by superoxide anion, limiting ROS production by the mitochondrial respiratory chain. This in turn decreases superoxide levels and UCP uncoupling activity via a feedback loop . Fatty aHow oncogenes and tumor suppressor genes may regulate UCP should be of great interest to study UCP in cancer. However, such evidence is rare, which deserves more research efforts, since knowing the basic biology of UCP regulation in cancer cells is essential for potential UCP-targeted cancer therapy. One important study utilized well-to-moderately differentiated primary breast tumor cell lines showed that SMAD was recruited to the UCP2 promoter region via the transforming growth factor-beta (TGFβ)/SMAD-dependent pathway . In cont5.p53, as a pivotal tumor suppressor, can cause apoptotic cell death in response to cellular stress stimuli  and the expression of viral or cellular oncogenes . Intereset al. demonstrated that UCP2 inhibited apoptosis of colon cancer cells by suppressing the phosphorylation of p53 within the transactivating domain via suppressing ROS [et al. had a different observation. They reported that high UCP2 expression in invasive breast ductal carcinoma was associated with high p53 nuclear expression [A few studies have suggested that this may indeed occur. Derdak sing ROS . Won et pression . Howeverpression . TherefoOur studies using the skin carcinogenesis model revealed that during early tumor promotion, the tumor suppressor p53 translocated to mitochondria and physically interacted with a primary antioxidant defense enzyme, manganese superoxide dismutase (MnSOD), leading to suppression of its superoxide scavenging activity, as well as, increases in ROS levels . Thus, iGiven these facts, we further examined the role of UCP2 in tumor promoter-induced p53 mitochondrial translocation and cell transformation. Downregulation of UCP2 expression using a transfection with specific siRNA to UCP2 induced translocation of a small amount of p53 to mitochondria, and also enhanced tumor promoter-induced p53 mitochondrial translocation whereas cell transformation was reduced . These dThe elevated levels of UCP2 in cancer cells may be a result of long-term selection during tumorigenesis, since any event that results in UCP2 upregulation could help cells escape from apoptosis mediated by the p53 signaling. Given the fact that mitochondrial uncoupling could cause dissipation of the mitochondrial potential, a decrease in mitochondrial ROS, and a reduction in p53’s response to oxidative stress, it is reasonable to propose that mitochondrial uncoupling may provide cancer cells with a prosurvival advantage via suppressing the p53 mediated apoptosis pathway.6.It has been suggested that dysregulation of UCP is involved in the pathogenesis of not only diabetes and obesity, but also neurodegenerative disease, atherosclerosis, and cancer. Among these diseases, it is very likely that UCP can protect against neurodegeneration, but exacerbate the progression of the other diseases. In cancer, elevated UCP contributes not only to chemoresistance, but also to early transformation. Therefore, targeting UCP could serve as a promising approach for both cancer prevention and therapy.