Telomeres protect the integrity of information-carrying DNA by serving as caps on the terminal portions of chromosomes. Telomere length decreases with aging, and this contributes to cell senescence. Recent evidence supports that telomere length of leukocytes and skeletal muscle cells may be positively associated with healthy living and inversely correlated with the risk of several age-related diseases, including cancer, cardiovascular disease, obesity, diabetes, chronic pain, and stress. In observational studies, higher levels of physical activity or exercise are related to longer telomere lengths in various populations, and athletes tend to have longer telomere lengths than non-athletes. This relationship is particularly evident in older individuals, suggesting a role of physical activity in combating the typical age-induced decrements in telomere length. To date, a small number of exercise interventions have been executed to examine the potential influence of chronic exercise on telomere length, but these studies have not fully established such relationship. Several potential mechanisms through which physical activity or exercise could affect telomere length are discussed, including changes in telomerase activity, oxidative stress, inflammation, and decreased skeletal muscle satellite cell content. Future research is needed to mechanistically examine the effects of various modalities of exercise on telomere length in middle-aged and older adults, as well as in specific clinical populations. Telomeres protect the integrity of information-carrying DNA throughout cell cycle by serving as specialized DNA caps on chromosomes. During successive cellular divisions, telomeres prevent base pair loss of chromosomal DNA. Over time, telomere length decreases until the telomere becomes too short for the cell to divide, resulting in cellular senescence. This scenario is known as the end replication problem and describes the inability of DNA polymerase to replicate the terminal ends of the chromosomes (i.e. the telomeres) . TelomerIt is suggested that telomere length may be a potential cellular marker for biological aging, as demonstrated by an inverse relationship between age and telomere length . AlthougCurrent evidence supports the idea that telomere length is associated with a number of chronic conditions including dyslipidemia , hyperteIt is well known that physical activity is associated with healthy aging and reduced risk for a number of chronic conditions , though Based on the significance of telomere length in aging and the need to understand the potential association with physical activity, the purpose of this systematic review is to investigate whether physical activity and exercise influence telomere length and to discuss possible mechanisms of action.A PubMed literature search was performed in accordance with the PRISMA statement . The seaObservational studies examining the potential relationship between physical activity/exercise and telomere length in skeletal muscle cells and leukocytes are summarized in Table st quartile of exercise energy expenditure  and 4th quartile  had shorter peripheral blood mononuclear cell (PBMC) telomere length than those in the 2nd quartile . There was no difference in PBMC telomere length between the 2nd and 3rd quartiles, suggesting that moderate levels of physical activity may be beneficial in protecting telomere shortening. Interestingly, telomerase activity in PBMC remains similar in all quartiles of physical activity [et al. in which they observed that, compared to sedentary adults, active individuals participating in long-distance running showed elevated telomerase activity, increased telomere-stabilizing proteins, down-regulated cell-cycle inhibitors, and decreased telomere erosion in the circulating leukocytes [vs. leukocytes) and a discrepancy in sample sizes between the two studies (69 vs.104 subjects). More recent studies support that there is a clear dose-response relationship between movement-based behaviors and telomere length. Accordingly, greater vigorous physical activity is associated with a longer telomere length, as assessed in leukocytes and salivary DNA , measured cell types, and self-reported activities Table  and 2. Oactivity . This isukocytes . The conThree independent studies evaluated the effect of moderate to vigorous aerobic exercise on skeletal muscle or leukocyte telomere length in young and older adults , 47, 48.2 max) [2 max across groups [Several researchers investigated additional factors that may correlate with telomere length. Studies showing differences between older athletes and non-athletes in their skeletal muscle or leukocyte telomere length also reported positive correlations between telomere length and aerobic fitness (VO2 max) , 47. In s groups . This las groups . Howevers groups , 49.et al. examined relative leukocyte telomere length in 4,576 individuals on two occasions, with 10 years between each measurement [The evidence indicates that individuals engaged in multiple healthy behaviors are more likely to maintain telomere length. Telomere length is likely altered by a variety of factors, and some may not yet be known, which could explain the mixed findings between physical activity/exercise and telomere length. For instance, a recent study examining a nationally representative US population showed that leukocyte telomere length moderately correlates with known cardiovascular risk factors such as blood glucose levels, thus serving as a potential predictor of cardiovascular diseases. Specifically, the percentage of body fat demonstrated a strong negative correlation with leukocyte telomere length regardless of age and health behaviors  . Psycholsurement . The ressurement .et al. showed that leukocyte telomere length is positively associated with baseline VO2 max and inversely associated with age, while unassociated with BMI and percent body fat [et al. reported a strong negative correlation between telomere length and sitting time in the exercise training group but not in the sedentary control group, suggesting a greater importance of reduced sitting time than increased exercise time in telomere maintenance [There are three reports of randomized controlled intervention study that investigated the effects of exercise on leukocyte telomere length, as summarized in Table body fat . Howeverntenance .In a small quasi-experiment study, investigators studied the effects of acute exercise on leukocyte recruitment and subsequent mean telomere length in young and old subjects . After aBoth chronic diseases and the aging process are known to be associated with decreased telomere length, but it may be possible to preserve telomere length with physical exercise. Although the existing literature does not provide adequate experimental evidence to fully establish the mechanistic relationship between physical exercise and telomere length, several potential mechanisms exist to explain how physical activity may affect telomere length: telomerase activity, oxidative stress, inflammation, and skeletal muscle satellite cell content Figure .et al. reported that from age of 4 to 39 years, there is a progressive decrease in both telomere length and telomerase activity. However, in individuals aged 40 or over, where telomere length continued to progressively decrease, 65% of individuals had a stable but low telomerase activity, and the remaining 35% of individuals had no telomerase activity [et al. compared track and field athletes to sedentary adults, and found an up-regulation of telomeric repeat-binding factor 2 (TRF2), a protein that plays a role in protecting telomeres from shortening, in the athletes (both young and middle-aged) [While telomere length shows a progressive, age-related shortening, telomerase demonstrated a biphasic pattern of expression. Iwama activity . Physicale-aged) . In younet al. determined that several oxidative-stress genes are linked with both telomere attrition and biological aging [Excessive reactive oxygen species (ROS) production can cause oxidative stress in cells, tissues or organs –74, leadal aging . Moreoveal aging . Correlaal aging , 28. Fural aging . In factal aging . It has al aging . Althougal aging . Therefoal aging . Diets tal aging .The attenuation of chronic inflammation provides another potential mechanism for the protective effects of exercise and physical activity on telomeres. For instance, chronic systemic inflammation elevates white blood cell (WBC) turnover, which increases telomere attrition rate . With inet al. recently described the complex interrelationship between aging, inflammation, and telomere length [Chronic obstructive pulmonary disease (COPD) patients have been reported to possess shorter mean leukocyte telomere length than healthy controls, which may be linked with chronic inflammation status in these patients. Specifically, IL-6 levels were inversely correlated with telomere length in COPD . Zhang ee length . They dre length , 89.Satellite cells are skeletal muscle cell precursors which can regenerate muscle cells or additional satellite cells in response to muscle injury. After age 70, the number of satellite cells is reported to decrease . This coRecent observational and experimental studies reveal a possible relationship between physical activity/exercise and telomere length, although this is not entirely clear from the present body of literature. Evidence has shown that physical activity decreases chronic inflammation and oxidative stress, especially in older, obese individuals, and may thus reduce telomere shortening observed with aging . TherefoIn addition to physical activity, weight loss itself may reverse telomere attrition. Since age 50, women who were overweight or obese and later lost weight to be normal, or women who sustained a constant weight within 5%, has shown greater leukocyte telomere length than those who gained weight after age 50 . By mainTelomere length has inverse associations with chronic conditions . Physical activity and exercise may be beneficial for telomere length maintenance in both healthy and chronically ill middle-aged and older adults. Telomere length is not only a marker of aging, but also relates to the ability to protect DNA from damage and the associated consequences. Individuals living with chronic conditions are more likely to be sedentary and experience functional limitations and disability. Physical activity and exercise may have both protective and restorative effects, and as such, show great potential to improve well-being and increase longevity. However, more interventional studies, especially those with longer-term exercise, are needed to confirm specific effects of various doses and intensities of exercise training on telomere length, particularly in middle-aged and older adults who are at increased risk for chronic diseases associated with inflammation and oxidative stress.


In the dry Swiss Rhone Valley, Scots pine forests have experienced increased mortality in recent years. It has commonly been assumed that drought events and bark beetles fostered the decline, however, whether bark beetle outbreaks increased in recent years and whether they can be linked to drought stress or increasing temperature has never been studied.In our study, we correlated time series of drought indices from long-term climate stations, 11-year mortality trends from a long-term research plot, and mortality probabilities modeled from tree rings  with documented occurrences of various bark beetle species and a buprestid beetle, using regional Forest Service reports from 1902 to 2003 and advisory cases of the Swiss Forest Protection Service (SFPS) from 1984 to 2005. We compared the historical findings with measured beetle emergence from a 4-year tree felling and breeding chamber experiment.The documented beetle-related pine mortality cases increased dramatically in the 1990s, both in the forest reports and the advisory cases. The incidents of beetle-related pine mortality correlated positively with spring and summer temperature, and with the tree-ring-based mortality index, but not with the drought index. The number of advisory cases, on the other hand, correlated slightly with summer drought index and temperature, but very highly with tree–ring—based mortality index. The tree-ring-based mortality index and observed tree mortality increased in years following drought. This was confirmed by the beetle emergences from felled trees. Following dry summers, more than twice as many trees were colonized by beetles than following wet summers.We conclude that increased temperatures in the Swiss Rhone Valley have likely weakened Scots pines and favored phloeophagous beetle population growth. Beetles contributed to the increased pine mortality following summer drought. Among the factors not addressed in this study, changed forest use may have also contributed to increased beetle populations and Scots pine mortality, whereas air pollution seems to be of lesser importance.


Selenium (Se) is an essential micronutrient for human health. Se deficiency affects hundreds of millions of people worldwide, particularly in developing countries, and there is increasing awareness that suboptimal supply of Se can also negatively affect human health. Selenium enters the diet primarily through the ingestion of plant and animal products. Although, plants are not dependent on Se they take it up from the soil through the sulphur (S) uptake and assimilation pathways. Therefore, geographic differences in the availability of soil Se and agricultural practices have a profound influence on the Se content of many foods, and there are increasing efforts to biofortify crop plants with Se. Plants from the Brassicales are of particular interest as they accumulate and synthesize Se into forms with additional health benefits, such as methylselenocysteine (MeSeCys). The Brassicaceae are also well-known to produce the glucosinolates; S-containing compounds with demonstrated human health value. Furthermore, the recent discovery of the selenoglucosinolates in the Brassicaceae raises questions regarding their potential bioefficacy. In this review we focus on Se uptake and metabolism in the Brassicaceae in the context of human health, particularly cancer prevention and immunity. We investigate the close relationship between Se and S metabolism in this plant family, with particular emphasis on the selenoglucosinolates, and consider the methodologies available for identifying and quantifying further novel Se-containing compounds in plants. Finally, we summarize the research of multiple groups investigating biofortification of the Brassicaceae and discuss which approaches might be most successful for supplying Se deficient populations in the future. Awareness of malnutrition, e.g., deficiencies in iron, iodine, vitamin A, and zinc, in the developing world is high, but micronutrient deficiency is rarely discussed in developed countries. This reduced awareness is surprising, especially since micronutrient deficiency or suboptimal supply of the essential micronutrient selenium (Se) is seen in several developed countries such as New Zealand to Thomson, , Austral−1. Indirectly, Se deficiency results in loss of protective anti-cancerogenic effects through reduced expression of antioxidant selenoproteins and reduced availability of several seleno-compounds , As Se is an essential micronutrient, under-supply has direct and indirect consequences for human health. Direct disorders include a destabilized immune system, hypothyroidism and cardiomyopathy Whanger, . PatholoWhanger, . The dieBrassica species  there was a poor correlation between Se uptake and Se soil content grown on a landfill , 80% of the Se remained in soils , kale , cabbage (B. oleracea Capitata Group) and Chinese cabbage , they reported the detection of several non-typical glucosinolates. They postulated methylseleno-indole glucosinolates and glucosinolates acylated with methylselenoacetic acid or with methylselenosinapinic acid at position 6' of the thioglucose moiety. Typically, acylated glucosinolates are only found in seeds and not in other plant parts  from selenoamino acids may also result in the production of further Se-containing metabolites including selenosugars, selenosinapine, and selenourea derivatives as shown in mustard seeds  provides quantification by measuring the light emitted from excited ions and atoms at characteristic wavelengths. Excited ions and atoms are formed by reaction of the sample in an inductively-coupled plasma (ICP) and detected by atomic emission. For Se, the detection limit is relatively high due to the poor emission intensity of Se compared to other elements. The primary detection wavelength is 196.026 nm with small interference with iron complicating the analysis of Se in iron rich samples . The relatively large mass deficiency, coupled with a distinctive isotope pattern  or directly as selenate. To date mainly incorporation of Se via SeMet has been observed  pseudomolecular ion , grown on naturally Se-rich soil. These identifications are based on high resolution LC-MS, however, the exact structures of these compounds cannot be determined from the mass spectral data alone.The increasing power of very high mass resolution LC-MS instruments to identify Se-containing metabolites in e et al. . Selenos2Se), dimethyldiselenide (Me2Se2) and dimethylthioselenide (MeSSeMe) in transgenic tobacco  containing beta hydrogens (R2CHC(SeO)R2) and selenones  are thermally unstable above room temperatures and are not suitable for standard GC-MS analysis. For highly volatile selenides such as Me2Se and Me2Se2, headspace analysis using highly adsorbent graphite based SPME phases such as CarboxenTM or CarboxenTM-PDMS hybrid fibers  is very useful for the identification of Se-containing metabolites, however finding minor Se containing species in complex plant extracts is tedious and has disadvantages such as the oxidation of small Se-molecules and lower sensitivity engendered by the complex isotope distribution of Se. A robust approach, which should be also applied for the analysis of A. fistulosum; Shah et al., Allium species . H2Se is the key metabolite formed from inorganic sodium selenite via selenodiglutathione through reduction by thiols and NADPH-dependent reductases and released from SeCys by a lyase-dependent reaction , organic hydroperoxides, and phospholipid hydroperoxides (only GPx4) using reduced glutathione as co-substrate  upon reaction with and oxidation of thiols. These compounds are therefore termed as redox-active Se compounds  and in vivo (chickens) compared to their un-selenated forms . There does appear to be some opposing effects of Se and isothiocyanates. The former is more commonly related to immune enhancing effects (Hoffmann and Berry, ex vivo indicated that the overriding effect was immune-stimulatory (Bentley-Hewitt et al., −1 to 110 ± 3 ng ml−1 after Se-enriched broccoli consumption, along with cytokines interleukin-2 and interleukin-4 production from participants' peripheral blood mononuclear cells when stimulated with phorbol 12-myristate 13-acetate and ionomycin, whilst no increases were observed following consumption of control broccoli (Figure in vitro showed anti-inflammatory effects (Peñas et al., in vivo and in vitro studies. It appears that the anti-inflammatory effects of Brassica phytochemicals may predominate in vitro. This does not explain why Se supplementation of Brassicas in Peñas et al. (Brassica extracts that were not supplemented with Se. However, it suggests that digestion, absorption, Se status, modification of the bioactive compounds by other cell types and/or the complexity of cellular cross talk in vivo may have a great influence on how immune cells respond to the bioactive compounds in Se-enriched Brassicas. The in vitro study by Peñas et al. (in vivo e.g., seleno amino acids. To start to elucidate the reasons for the discrepancy between in vitro and in vivo results, one could use in vitro digestion of Se-enriched Brassica prior to exposure to cells in vitro and extract material to include seleno amino acids. Previous research, utilized in vitro digestion of Se-enriched broccoli before testing with colon cancer cells and found a reduction in H2O2 production, however no inflammatory markers were measured (Tsai et al., Brassicas on immune respones particulary human in vivo data is limited.Substantial evidence exists that both Se and i Figure . Additioi Figure . In conts et al.  have enhs et al.  includesWe still do not know the impact of all the Se-containing bioactives, which may be driving the immune response. Research should focus on whether naturally occuring selenoglucosinolate hydrolysis products and amino acids are more bioactive than the S-containing analogs. Additionally, research is required to ascertain whether modification to immune signals, such as increased levels of cytokines, results in a more robust immune response in humans.Selenium deficiency or suboptimal Se intake is still regarded as a major health problem for about one billion people worldwide, while an even larger number may consume less Se than required for optimal protection against cancer, cardiovascular diseases, and severe infectious diseases (Haug et al., Brassica vegetables are consumed regularly worldwide. Thus, Se-biofortification of Brassica crops is an important biotechnological tool that can be used to benefit of Se nutrition in humans. Furthermore, beside the general Se-metabolites, such as seleno-proteins and seleno aminoacids, which can be found in most plant species, Brassicales also contain specialized Se-containing compounds with health benefiting properties such as MeSeCys. Also, the recent discovery of significant amounts of the selenoglucosinolates in biofortified broccoli is encouraging as it opens a further avenue for the production of potentially health promoting compounds in this genera. However, so far no investigations have been conducted on the biosynthesis of selenoglucosinolates or on their bioefficacy in human health, which would be of particular interest due to the distinct protective potential of their potential hydrolysis products.Due to their distinct biodiversity, Increasing Se uptake by the Brassicales is best achieved via hydroponic means, where Se exposure can be carefully controlled and uptake maximized. For crops already grown using hydroponic or similar systems, such as drip lines, Se biofortification should be relatively easy to implement. However, alongside this it will be critical to consider issues relating to Se toxicity, as even Brassicales of the same species can have variable Se uptake rates. This means that a “one size fits all” approach cannot be implemented and biofortificaiton regimes will have to be established for each crop and over the entire plant growth period in order to produce material with a known and stable Se content. Despite this, Se fertilization strategies have been successfully achieved in Finland to counter Se deficiency and should be investigated by other countries where the malnutrition of Se effects the human health of their population.Although, critical Se intake levels have already been determined with respect to Se undernourishment, we still need to understand the modes of action of individual Se-compounds in human metabolism before recommendations can be made for specific diseases. So far, no daily intake recommendation is established regarding the prevention of chronic diseases such as cancer or the maintenance of a well-regulated immune system. Moreover, further investigations are required to better understand the bioavailability and molecular effects of the selenoglucosinolates before determining their effective concentration for protection against chronic diseases. Achieving these goals will further establish the role Se plays in supporting human health, particularly through members of the Brassicales.MW: Corresponding author; MS, FSH, DS, MM: Section Selenium in Brassicales; SB and DR: Section Instrumental Approaches for Detecting, Measuring, and Monitoring Selenium and Its Metabolites within Brassica Species; AK, KBH, FSH, MW: Section Selenoglucosiolates for Human Nutrition.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.


An enhanced thrombotic environment and premature atherosclerosis are key factors for the increased cardiovascular risk in diabetes. The occlusive vascular thrombus, formed secondary to interactions between platelets and coagulation proteins, is composed of a skeleton of fibrin fibres with cellular elements embedded in this network. Diabetes is characterised by quantitative and qualitative changes in coagulation proteins, which collectively increase resistance to fibrinolysis, consequently augmenting thrombosis risk. Current long-term therapies to prevent arterial occlusion in diabetes are focussed on anti-platelet agents, a strategy that fails to address the contribution of coagulation proteins to the enhanced thrombotic milieu. Moreover, antiplatelet treatment is associated with bleeding complications, particularly with newer agents and more aggressive combination therapies, questioning the safety of this approach. Therefore, to safely control thrombosis risk in diabetes, an alternative approach is required with the fibrin network representing a credible therapeutic target. In the current review, we address diabetes-specific mechanistic pathways responsible for hypofibrinolysis including the role of clot structure, defects in the fibrinolytic system and increased incorporation of anti-fibrinolytic proteins into the clot. Future anti-thrombotic therapeutic options are discussed with special emphasis on the potential advantages of modulating incorporation of the anti-fibrinolytic proteins into fibrin networks. This latter approach carries theoretical advantages, including specificity for diabetes, ability to target a particular protein with a possible favourable risk of bleeding. The development of alternative treatment strategies to better control residual thrombosis risk in diabetes will help to reduce vascular events, which remain the main cause of mortality in this condition. Despite advances in therapy, cardiovascular disease (CVD) remains the main cause of morbidity and mortality in individuals with diabetes. The risk of vascular complications is doubled in diabetes, resulting in significant reduction in life expectancy [Diabetes is becoming the epidemic of the twenty first century with more than 1.5 million deaths worldwide directly attributed to this condition in 2012  binds factor VII (FVII), promoting proteolysis and activation to FVIIa. The TF/FVIIa complex activates FIX and FX resulting in the generation of FIXa and FXa, and the latter subsequently associates with cofactor FVa to form a prothrombinase complex, which converts prothrombin into thrombin . The genNormal physiology ensures a balance between fibrin clot formation and lysis in order to prevent widespread vascular occlusion or excessive bleeding. Plasmin is the enzyme responsible for fibrin breakdown (fibrinolysis) and is generated from plasminogen through the action of the serine protease tissue plasminogen activator (tPA), a product of endothelial cells . The binSeveral proteins prevent unregulated plasmin or plasminogen activator activity, thereby inhibiting excessive clot lysis. Plasminogen activator inhibitor PAI-1) is produced by endothelial cells, platelets and adipose tissue. Like other serpins (serine protease inhibitors), PAI-1 forms a stable 1:1 complex with tPA, thereby inhibiting its action . Plasmin is produIndividuals with diabetes suffer from premature atherosclerosis, a key contributing factor to increased cardiovascular risk in this population. This is treated in the acute stage with revascularisation followed by preventative therapy in the long-term, including control of blood pressure, lipid and glucose levels. To control thrombosis risk following an acute vascular event, dual antiplatelet therapy is usually used , 32, wheThe risk of aggressive anti-thrombotic therapy is increased bleeding which may negate any beneficial effect of such therapy. A prime example is the TRITON study that has shown that the combination of prasugrel and aspirin is superior to clopidogrel and aspirin at reducing vascular ischaemia following myocardial infarction but there was an increased risk of bleeding with the former combination making widespread clinical use problematic . InteresReducing the thrombotic environment in arterial occlusive disease relies mainly on antiplatelet therapy, using the cyclooxygenase inhibitor aspirin with or without P2Y12 antagonists clopidogrel, prasugrel or ticagrelor. While all anti-platelet agents are likely to alter clot structure through indirect effects on platelet-dependent thrombin formation, aspirin is unique amongst anti-platelet agents as it directly alters clot structure and enhances fibrinolysis, related, at least in part, to acetylation of fibrinogen. This makes aspirin an agent that independently targets both the cellular and protein phase of coagulation –37. It rAntithrombotic therapy in diabetes can be divided into primary prevention, which includes individuals without a previous history of vascular ischaemia, and secondary prevention in those who have had a vascular occlusive event. Until relatively recently, aspirin has been used for primary vascular protection in diabetes but a number of studies suggest a lack of significant success with such an approach . TherefoIn individuals sustaining a coronary event, current guidelines advocate the use of dual antiplatelet therapy (DAT) to reduce thrombosis risk for a period of 1 year, followed by lifelong antiplatelet monotherapy . InteresOne year after the vascular event, P2Y12 inhibitor therapy is usually stopped and patients continue on aspirin treatment. However, this approach is questionable in diabetes as the efficacy of aspirin appears to be compromised, secondary to a number of factors including increased platelet protein glycation and higher platelet turnover in diabetes . ImproviOne striking observation of clinical studies aiming to reduce atherothrombosis risk is the concentration on antiplatelet therapy, generally ignoring the contribution of the protein arm of coagulation to the vascular ischaemic event. Early work has shown that warfarin, an agent that inhibits the production of various coagulation proteins, can indeed improve outcome after arterial occlusion. However this agent is associated with a narrow safe therapeutic window and the combination with antiplatelet agents greatly increases bleeding risk . More moFXII inhibitors offer a potential alternative antithrombotic strategy in diabetes that do not carry the bleeding risk associated with the aforementioned agents. FXII initiates the intrinsic coagulation pathway, however, FXII deficiency in mice is not associated with increased bleeding and offers protection against thrombosis. FXII inhibition may therefore be useful in instances involving activation of the intrinsic pathway (such as stent thrombosis) but may not be appropriate if the underlying pathology is plaque rupture and future research in this area is certainly needed .It should be remembered that acute fibrinolytic therapy has been successfully employed as a treatment for acute vascular occlusion and therefore strategies that facilitate clot lysis on a long-term basis have the potential to control the risk of atherothrombosis, particularly in conditions characterised by impaired fibrinolysis.Although the current review concentrates on macrovascular complications in diabetes, studies suggest that hypofibrinolysis is also associated with microvascular complications –51. TherFactors involved in increased thrombosis risk in diabetes and current anti-thrombotic strategies are summarised in Table Hypofibrinolysis is a key abnormality in individuals with diabetes and therefore modulation of this pathological process may offer therapeutic benefits. The main factors influencing fibrinolysis in diabetes include altered fibrin network structure, increased incorporation of antifibrinolytic proteins into the clot and compromised activity of the fibrinolytic system.Compact fibrin networks with densely packed thin fibres are associated with increased risk of cardiovascular events –57, whicIndividuals with type 1 and type 2 diabetes have prothrombotic compact fibrin networks which correlate with glycaemic control measured as HbA1c –66. To cA number of mechanisms are responsible for altered clot structure in diabetes including quantitative and qualitative changes in coagulation factors as well as altered thrombin generation.Previous studies have observed a relationship between elevated plasma levels of fibrinogen and the risk of CVD –72. EvidPlasma levels of fibrinogen in diabetes subjects has been a focus of much research, however, results are not always in agreement. The majority of, but not all, studies found elevated plasma fibrinogen levels in individuals with T2DM, and some reported higher levels in individuals with T1DM (summarised in ). The RoTaken together, elevated fibrinogen levels appear to contribute to the compact fibrin networks observed in diabetes, at least in some patients.Protein glycation is a post translational modification, the extent of which is determined by ambient glucose levels and the duration of protein exposure. Glucose is able to bind non-enzymatically to fibrinogen by condensation of the carbonyl groups with amino groups on the fibrinogen molecule. Elevated glucose levels increase plasma protein glycation, including that of fibrinogen –78, withFibrinogen glycation in subjects with diabetes is modulated by improving glycaemic control , 78, 82.It should be noted, however, that glycation is not the only post-translational modification of fibrinogen in diabetes . DiabeteFibrin fibre diameter decreases with increasing thrombin levels. Low thrombin concentrations  produce clots composed of loosely woven, thick fibres, whilst clots formed at high thrombin tend to be made up of thin, tightly packed fibrin strands which are relatively resistant to lysis , 88. TheElevated thrombin generation has been reported in subjects with T2DM , 91–93. The mechanisms for enhanced thrombin generation in individuals with diabetes are not entirely clear. A study involving T1DM and T2DM patients showed that high levels of coagulation factors II,V,VII,VIII and X, coupled with low levels of anticoagulation factor protein C in diabetes are likely to be factors contributing to enhanced thrombin generation . In anotIt is well established that low grade inflammation predisposes to atherothrombosis and a number of pathways have been implicated describing interactions between inflammatory and thrombotic molecules, reviewed elsewhere –100. ForA potentially important but overlooked diabetes-related mechanism for hypofibrinolysis is increased incorporation of antifibrinolytic proteins into the clots, specifically complement C3 and PI.Until recently, complement C3 was regarded as an inflammatory protein, but evidence suggests key interactions between the complement and coagulation systems . Two stuC3 binding/cross-linking into the fibrin network increases resistance of the clot to lysis . FurtherThe mechanisms for C3-induced compromise in clot lysis are not entirely clear but one may involve C3 interference with tPA and/or plasminogen binding sites on the fibrin network. Alternatively, C3 may affect the availability of plasmin to act on fibrin fibres, as C3 is a known substrate for plasmin . FinallyPlasmin inhibitor  belongs to the serpin superfamily of proteins. PI is a key protein in blood haemostasis, as evidenced by the disorders caused by homozygous deficiency or by protein over-production. Congenital deficiency of PI results in a severe bleeding disorder , 112, whAgren et al. found increased incorporation of PI into the fibrin network in individuals with T1DM, although these individuals exhibited a paradoxical reduction in clot lysis time, which the authors attributed to reduced PAI-1/fibrinogen levels . An earlA number of abnormalities in the fibrinolytic system have been reported in diabetes including elevated PAI-1 and TAFI levels, and deranged plasminogen function.There is some evidence to suggest that antifibrinolytic protein TAFI is upregulated in diabetes, which could contribute to the hypofibrinolytic state. Elevated plasma levels of TAFI are associated with increased risk of cardiovascular diseases –120. In PAI-1 levels are associated with increased risk of CVD  with eleThere is also an association of elevated PAI-1 levels and abdominal obesity as adipose tissue expresses PAI-1 and represents an important source of plasma PAI-1 in obese subjects –133. ThiThe hyperglycaemic environment in diabetes is responsible for glycation of plasminogen, which compromises protein function. Plasminogen purified from plasma of individuals with T1DM has a decreased rate of conversion to plasmin, and the plasmin generated has altered proteolytic activity, resulting in impaired fibrinolysis. Moreover, improving glycaemic control partially restores plasminogen conversion to plasmin and the activity of the enzyme, further emphasising the importance of maintaining good glycaemic control to reduce thrombosis risk. Interestingly, only a modest improvement in glycaemic control is enough to result in significant improvement in plasmin activity . This suTherefore, hyperglycaemia enhances thrombosis potential by increasing plasma levels of pro-coagulant and antifibrinolytic factors as well as modulating protein activity by introducing post-translational modifications in coagulation proteins. It should be noted, however, that not every single factor that modulates fibrinolysis has been comprehensively studied in diabetes, particularly in relation to the various subgroups of patients. Therefore, in addition to developing agents that target the large number of anti-fibrinolytic proteins, future studies are required to understand the most relevant anti-fibrinolytic strategy in the different subgroups of diabetes patients. This may indeed involve targeting multiple proteins/pathways, particularly in the higher risk groups, to maximise clinical benefit.A summary of the mechanisms involved in hypofibrinolysis in diabetes is provided in Fig. The potential role of various hypoglycaemic agents in thrombosis risk is beyond the scope of this review and will therefore concentrate on the effect of glycaemia per se on thrombosis potential.From the evidence presented above, it is clear that hyperglycaemia results in a prothrombotic and hypofibrinolytic environment. Moreover, relatively modest improvement in glycaemia appears to have a significant effect on fibrin network structure and/or resistance to lysis. It should be noted, however, that overtreatment of hyperglycaemia, and precipitation of hypoglycaemia, can also be prothrombotic . StudiesTaken together, the above findings may offer mechanistic explanations for the disappointing clinical outcome trials investigating the role for tight glycaemic control in reduction of vascular ischaemic events in diabetes . It is pFrom the practical point of view, using agents that are less likely to cause hypoglycaemia may have the advantage of reducing the thrombotic environment in diabetes and protecting against vascular ischaemic events. We have limited evidence to suggest that agents that do not cause hypoglycaemia, such as metformin, pioglitazone, empagliflozin and liraglutide are associated with favourable cardiovascular profile –144. In Given that diabetes is associated with increased plasma levels of PAI-1 and TAFI, and increased incorporation of PI and C3 into the clot, targeting these proteins may alleviate the hypofibrinolytic environment, consequently decreasing atherothrombotic risk.TAFI circulates in an inactive zymogen form and is activated by thrombin, plasmin, or the thrombin-thrombomodulin complex. Activated TAFI cleaves C-terminal lysine residues from partially degraded fibrin, which are critical for the binding of plasminogen and as a result, plasmin generation is reduced .Camilidae family which have advantageous properties such as low immunogenicity and high affinity, solubility and stability [Inhibition of TAFI was considered as a therapeutic strategy in thrombotic disorders but only a limited number of drug candidates have made it to clinical trials, which were then discontinued . Mtability . Two nantability . AlthougPAI-1 is present in an active and a latent state in vivo with the active form having high affinity for vitronectin, which binds PAI-1 in the blood . PAI-1 hSmall molecule inhibitors of PAI-1 synthesis include synthetic and natural products such as niacin, fibrates and butadiene derivatives (reviewed in ). HoweveThere have been numerous attempts at inhibiting PAI-1 with antibodies, reviewed elsewhere , 156 butA number of groups have employed different methods for identification of PAI-1 inhibitors, including high throughput and in silico screening, reviewed extensively elsewhere , 160. UsOther small molecule inhibitors have been identified, such as ZK4044 which binds directly to PAI-1, preventing its interaction with target proteases , WAY-140The reported increased incorporation of PI into fibrin clots in diabetes renders this protein a potential diabetes-specific therapeutic target. Numerous attempts at targeting PI have been made, including the use of antibodies and mutant forms of the protein.2 fragments reduced circulating PI levels and led to an acceleration of thrombolysis by enhancing fibrinolytic activity [Kumada et al. reported that repeated injection of polyclonal anti-PI F(ab’)activity . A more activity . Similaractivity . An exteactivity .Arginine residue 364 is the main reactive site on PI, which forms a covalent bond with the active site serine in plasmin, resulting in an inactive protease-inhibitor complex , 172. LeThe N-terminal region of PI is crucial for cross-linking to fibrinogen , 176, whOthers attempted to use the C-terminus of PI as a means of enhancing fibrinolysis. A 26 amino acid peptide composed of the carboxy terminal region of PI was found to enhance in vitro fibrin clot lysis by increasing conversion of plasminogen to plasmin approximately fivefold . SimilarPlasmin inhibitor is present in two forms in human plasma, a 464-residue protein with methionine at the N-terminus (Met-PI) and a truncated 452-residue protein with asparagine at its N-terminus (Asn-PI). Human plasma contains around 30% Met-PI and 70% Asn-PI form. Asn-PI, formed from Met-PI by the action of antiplasmin cleaving enzyme (APCE), is more rapidly cross-linked to fibrin by FXIIIa –184. APCDespite promising preliminary data, none of these approaches of PI-related therapeutic targets have been clinically adopted, indicating that translating in vitro findings, and even animal work, into application in man is difficult and more complex than initially envisaged.Therapeutic intervention in the complement system has been recognised as a potential strategy for the treatment of a series of inflammatory and autoimmune diseases , 187 whiPotential future strategies to enhance the fibrinolytic process, and reduce thrombosis risk in diabetes are summarised in Table Despite advances in treatment, vascular mortality in patients with diabetes remains unacceptably high, which is partly related to the enhanced thrombotic environment that is not fully controlled with current anti-platelet therapies. Rather than developing more effective anti-platelet agents, which runs the risk of bleeding complications, an alternative approach is to target the hypofibrinolytic state in diabetes, a key abnormality in this condition. Therefore, understanding the mechanisms for increased fibrin-related thrombosis risk in diabetes is key to develop effective and safe novel antithrombotic therapies.Diabetes is associated with both quantitative and qualitative changes in clotting factors resulting in denser fibrin networks that are more difficult to lyse. In addition to changes in clot structure, diabetes directly impairs the function of the fibrinolytic system and shows increased incorporation of anti-fibrinolytic proteins into the clot. More specifically, recent studies have shown increased incorporation of PI and complement C3 into diabetes clots, which compromise fibrin clot lysis. The increased incorporation of anti-fibrinolytic proteins into fibrin networks represents a novel diabetes-specific mechanistic pathway that may be a target for a new generation of anti-thrombotic agents.In summary, targeting alternative and diabetes-specific thrombotic pathways may be the best approach to reduce the residual thrombosis risk in diabetes and improve vascular outcome in this population.


Aminoglycoside antibiotics are used as prophylaxis, or urgent treatment, for many life-threatening bacterial infections, including tuberculosis, sepsis, respiratory infections in cystic fibrosis, complex urinary tract infections and endocarditis. Although aminoglycosides are clinically-essential antibiotics, the mechanisms underlying their selective toxicity to the kidney and inner ear continue to be unraveled despite more than 70 years of investigation. The following mechanisms each contribute to aminoglycoside-induced toxicity after systemic administration: (1) drug trafficking across endothelial and epithelial barrier layers; (2) sensory cell uptake of these drugs; and (3) disruption of intracellular physiological pathways. Specific factors can increase the risk of drug-induced toxicity, including sustained exposure to higher levels of ambient sound, and selected therapeutic agents such as loop diuretics and glycopeptides. Serious bacterial infections (requiring life-saving aminoglycoside treatment) induce systemic inflammatory responses that also potentiate the degree of ototoxicity and permanent hearing loss. We discuss prospective clinical strategies to protect auditory and vestibular function from aminoglycoside ototoxicity, including reduced cochlear or sensory cell uptake of aminoglycosides, and otoprotection by ameliorating intracellular cytotoxicity. Pseudomonas, Salmonella and Enterobacter species , and permanent hearing loss  and/or balance disorders (vestibulotoxicity). Aminoglycoside-induced vestibulotoxicity and/or cochleotoxicity occurs in as many as 20% of patients who received these drugs intravenously for multiple days  for clinical use in the United States. Of these, gentamicin, tobramycin, and amikacin are the most common parental agents. Gentamicin is often preferred because of its low cost and reliable bactericidal activity. It is administered systemically in neonatal intensive care units (NICU), mostly for prophylaxis in preterm infants, and discontinued once sepsis is ruled out <72 h. If sepsis is confirmed, treatment may continue for another 10–15 days. Tobramycin is primarily used for treating Almost all cells take up aminoglycosides, and most cells are able to clear these drugs from their cytoplasm relatively quickly, by mechanisms as yet undetermined, except for inner ear hair cells and renal proximal tubule cells which retain these drugs for extended periods of time  within the stria vascularis that generates an endocochlear potential (EP) as high as +100 mV. The stria vascularis is also a tight junction-coupled compartment and with the reticular lamina and Reissner’s membrane encloses the scala media, ensuring electrochemical separation of endolymph and perilymph  channels on the stereociliary membrane, enabling depolarizing transduction currents that trigger the release of the neurotransmitter glutamate, which in turn induces action potentials in the innervating afferent auditory neurons  to be resorbed in the ascending limb. As the tubule progresses into distal convoluted tubule, further cation reclamation  occurs as H+ is secreted into the remaining fluid, now recognized as urine that drains into the collecting duct and bladder prior to being voided.Drugs and toxins in the blood are excreted via ultra-filtration by the kidney. Renal arterial blood undergoes extravasation in kidney glomeruli, and the ultrafiltrate passes into the lumen of the proximal convoluted tubule Figure . EpithelThere are many physiological similarities between the cochlea and kidney, principally the active transport of electrolytes or nutrients, and consequently, water follows to maintain iso-osmolarity. Gene expression analysis has identified at least 36 genes that are significantly expressed in both cochlea and kidney  did not visibly affect hair cell uptake of intravenously-administered aminoglycosides. However, when aminoglycoside-laden artificial perilymph was perfused though the scala tympani, hair cell uptake of aminoglycosides over their basolateral membranes was markedly reduced compared to systemic delivery , associates with cubulin, a co-receptor, and when bound to aminoglycosides, the complex is endocytosed . The TRP vanilloid receptor 1, TRPV1, was identified using a number of channel modulators  cation channels that are also activated by osmotic swelling of cells, and chemically activated by 4α-phorbol 12,13-didecanoate  channels are inflammatory, irritant and oxidative stress sensors , resorbs 90% of lumenal glucose from renal ultra-filtrate in proximal tubules  are largely excluded by the blood-labyrinth barrier from inner ear tissues  in clinical settings. Preclinical models with systemic inflammation, induced by low doses of bacterial lipopolysaccharides displayed increased cochlear uptake of aminoglycosides, and enhanced levels of cochleotoxicity without altered serum drug levels  and caspase signaling cascades have been described elsewhere in detail  for the 28S rRNA than for 12S rRNA, a concentration readily reached in hair cells at clinically-relevant concentrations  receptors  properties of antioxidants in the organ of Corti explants is described elsewhere in this Research Topic  regulate this process. Aminoglycosides also hypo-acetylate histones, reducing transcription factor binding to DNA, causing decreased levels of gene expression , including HSP70, to promote hair cell survival against aminoglycoside ototoxicity  classes of broad-spectrum antibiotics. Aminoglycosides are also preferred due to their robust stability at ambient temperatures when used by itinerant healthcare providers in the field, and because of their bactericidal efficacy against bacteria resistant to other antibiotics. Increasing our understanding of aminoglycoside-induced (oto)toxicity requires greater insight into the mechanisms of cellular uptake kinetics, transcellular trafficking and intracellular disruption of physiological activities by aminoglycosides, especially in models that better mimic clinical settings such as exposure to higher levels of ambient sounds, co-therapeutics and/or inflammation that potentiate the degree of ototoxicity. Modifying dosing protocols, the structure of current aminoglycosides, and/or increased verification of candidate otoprotective agents could all enable aminoglycosides to be used more readily with reduced risks of lifelong ototoxicity in hospital.This review was conceived, written and edited by each of the authors .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.


Experimental renal cancer caused by ochratoxin A (OTA) in rats was first defined in the US National Toxicology Program (1989) and raised questions about any aetiological role in human urinary tract tumours. A review of histopathology in several rat kidney tumours from dietary OTA in recently described London studies, augmented by clinical immunohistochemistry for the first time for this mycotoxin, establishes their renal tubular cell origin. It had been assumed that the toxin might cause the human urothelial tumours associated with Balkan endemic nephropathy, but the present study could not support this. Comparison with a similar review of a metastasising renal tumour from a female rat of the NTP study consistently shows the kidney as the primary carcinogenic site for OTA. Morphological heterogeneity of these kidney tumours as epithelioid and/or sarcomatoid is revealed. Leiomyosarcoma was also diagnosed, and rhabdomyosarcoma differentiation was observed in the exceptionally aggressive NTP female tumour. The present pilot study involving immunohistochemistry indicates need for wider review of archived tumours for experimental evidence before formulating any epidemiological basis from a rat model for OTA’s relevance to idiopathic human renal cell carcinoma. Although the NTP study concluded that females are less sensitive to OTA than males, some female tumours still had heterogeneous morphology. Ochratoxin A (OTA) was first characterised in South Africa  but founMeanwhile, following the deductions of Krogh  that OTAThe present pilot study seeks for the first time to contribute to enhanced pathological definition of archived rat tumours, in response to chronic dietary exposure to OTA in the London studies ,13, alsoThe six malignant kidney tumours studied immunohistochemically  have varThe epithelioid component combines predominantly high-grade cytology with vesicular, nucleolated, mitotically active nuclei and abundant eosinophilic cytoplasm, which only occasionally appears clear. The tumour cells are arranged in a solid pattern, with nested, alveolar and tubular architecture; rarely noted are pseudopapillary structures. Sometimes, cystic or hyaline degeneration is seen. A common finding is necrosis; tumour lobules with central necrosis (comedo-like) are frequently present. The tumour invasion front is mostly of the pushing type .A sarcomatoid pattern is encountered in five cases (2–6) in various proportions, showing spindle cell morphology and exhibiting rhabdoid features in one case. The tumour invasion front is infiltrative; entrapped glomeruli and unaffected tubuli within the tumour mass are noted. The three cases with large sarcomatoid areas , have an aggressive biological behaviour; vascular and neural invasion  are seenTwo particularly interesting features have been noted, both of which independently imply a renal tubular cell origin of the tumour. In one tumour case, the infiltrative angulated tubuli and the desmoplastic reaction are suggestive of collecting-duct morphology. The second feature is found in another tumour case: a few well-defined and better-differentiated foci, revealed by immunohistochemistry, are present within the spindle cell pattern, supporting the renal cell origin of the tumour. Also supportive of a tubular cell origin is the fact that the adjacent renal parenchyma shows tubular intraepithelial dysplastic change in four cases, ranging from very focal and subtle to marked intraepithelial cytological atypia .Immunohistochemical profiles  point toThe fourth case is showing an extensive sarcomatoid pattern, lacking CD10 expression and showing vimentin and pancytokeratin MNF116 expression, with a weak focal CK7 reaction, mostly with luminal pattern. This vimentin-pancytokeratin co-expression could be suggestive of a sarcomatoid carcinoma; however, the dysplastic changes found nearby indicate a renal cell origin of the tumour .The most intriguing case (5), the only female rat in this immunohistochemical study, shows a large infiltrative tumour, involving the whole kidney, abutting and focally destroying the pelvic urothelial mucosa and invading the surrounding adipose tissue. This tumour shows high grade cytology with vesicular nucleolated nuclei, mitotically active, predominant epithelioid and spindle cell sarcomatoid morphology, including, focally, a small area with rhabdoid features, with abundant eosinophilic cytoplasm and eccentrically placed nuclei. A few well-defined foci are noted, crossing transversally the whole thickness of the kidney. These foci show a solid pattern and occasionally form tubular structures ; immunohThe last case (6) showing exclusively spindle cell morphology reveals intense and diffuse reaction for vimentin and smooth muscle actin , with onThe renal lesional background is represented in most cases by different stages of chronic progressive nephropathy including degenerative and regenerative aspects: vacuolar cytoplasmic degeneration; cortical subcapsular foci of small basophilic tubuli, some of them with thickened basement membrane; compensatory enlarged hypertrophic tubuli; cystically dilated tubuli or glomeruli with glomerular metaplasia of the parietal Bowman’s capsule ; casts, both hyaline and granular; variably sized cysts located within the medulla and lined by an attenuated epithelium (toxic effect or ageing related). Along with these features, regenerative tubule hyperplasia and even oncocytic hyperplasia  were ideAll cases show tubuli with various degrees of cytological atypia such as markedly enlarged , pleomorphic, hyperchromatic or vesicular nuclei, with prominent nucleoli. These tubuli are located at the cortico-medullary junction in the inner medulla and papilla. This type of atypia is either focal, for example, one isolated cell amongst the epithelial cells of the tubule, or sometimes the smaller cysts are lined entirely by such an atypical epithelium. However, this is not associated with proliferating activity.Tumour proliferation in these five cases ranges from large, poorly differentiated, metastasising tumour to small (0.5 mm) well-differentiated organ-confined lesions, which are even difficult to discriminate from atypical tubule hyperplasia. One case, carcass 1195 in , shows lTumour necrosis is seen in all but one case. Atypical tubule hyperplasia in one case is intimately associated with tumour mass, while in another case the lesion is away from the tumour. Variable degrees of focal-isolated karyomegaly are frequently seen, with morphology and distribution similar to that described in non-tumour kidney cases.Historically, the renal cell origin has been difficult to establish and only the present IHC could prove it. It is important to emphasize that these tumours are of renal tubular cell origin and not urothelial, as proven by immunohistochemistry and suggested by the renal intraepithelial dysplastic lesion. Morphologically the renal cell nature is not immediately obvious, the cytoplasm being rather eosinophilic and only focally clear, as expected from a conventional clear cell renal cell carcinoma. Also, the nuclei are showing prominent nucleoli, visible on low power, indicating a high nuclear grade (Fuhrman). Likewise, the frequent association of a spindle cell component (sometimes massive or even exclusive) potentially hinders the renal cell origin assessment. However, immunohistochemically, the co-expression of vimentin and CD10 and the lack of reaction for CK7, CKHMW and p63 are supportive of this origin. In normal kidney, CD10 is strongly expressed by the cytoplasm and membrane of podocytes and proximal tubular cell brush borders; in renal cell carcinoma CD10 is usually positive in clear cell  and papillary type renal cell carcinomas (RCC). In addition to this unequivocal immunohistochemical profile, an indirect criterion for non-urothelial cell origin is the fact that the sarcomatoid component association is rather uncommon for urothelial neoplasms; very few cases have been described in the literature . SarcomaThere is overall high grade cytology in both epithelioid and sarcomatoid areas, with high nuclear grade and necrosis; all these findings place these tumours in a poorly differentiated category. No classic conventional clear cell RCC picture has been identified; the epithelioid cells were mostly of eosinophilic granular cell type. When large amounts of spindle cell morphology are admixed, even merely associated or predominate, the degree of dedifferentiation is mirrored by the altered immune expression of CD10, which is lost. Instead, pan-CK expression occurs, which is variably co-expressed with vimentin, thus favouring the sarcomatoid nature of the tumour.RCC with sarcomatoid features is not currently recognized as an entity as a specific type of renal parenchymal carcinoma by the most recent 2016 WHO classification of renal tumours, mainly because sarcomatoid areas can be found in all histologic subtypes of RCC .Sarcomatoid RCC may arise out of a background of any histologic type of RCC as a manifestation of a final common dedifferentiation pathway. A pure sarcomatoid pattern is uncommon and, as recommended by the same previously mentioned guidelines, should be categorized as unclassified RCC (grade 4 unclassified carcinomas with a sarcomatoid component). The overwhelming sarcomatoid component found in these six tumours is striking because of its frequency and extent. OTA dose-factor difference does not seem to be crucial since, for example, within the same lot one tumour is organ-limited, small, nodular, well-demarcated, epithelioid and without spindle cell morphology, whilst the other one is a highly aggressive, metastasising tumour with sarcomatoid features.Rhabdoid differentiation in RCC refers to the development of neoplastic cells that morphologically resemble rhabdomyoblasts but differ in ultrastructural features and immunophenotype (being desmin negative). The similar immunophenotype of rhabdoid and non-rhabdoid foci supports the origin of the rhabdoid cells from renal cells. Areas of rhabdoid morphology do not represent metaplastic muscle differentiation . BearingCompletely different in the present study is case 6, showing a spindle cell proliferation which is positive diffusely for SMA and focally for desmin. This indicates a pure mesenchymal origin, suggestive of leiomyosarcoma.These poorly differentiated tumours have a highly aggressive course; they are usually very infiltrative, invading the surrounding local structures and metastasising at distant sites . The metastases have similar tumour morphology and are confirmed by the same immune profile. Consistently, all tumours at sites distant from a tumorous kidney were shown to have IHC profiles corresponding to the primary kidney neoplasm, confirming their metastatic status. This demonstrates the near-exclusive relationship between OTA and kidney concerning carcinogenicity in the rat, even after many months of exposure to the toxin.Dysplasia of tubular epithelium is probably a biologic precursor of at least some RCC, and it is also a strong support for the renal cell origin. In this study it was noticed within the adjacent renal parenchyma in four cases, ranging from mild to severe intraepithelial cytological atypia, similar to carcinoma in situ; interestingly, this lesion has been found occasionally in the contralateral kidney as well.Premalignant precursor lesions of RCC are not well characterized, but intratubular epithelial dysplasia defined morphologically by crowded, enlarged and pleomorphic nuclei has been described by other authors . HoweverAtypical tubule hyperplasia is a tubule-confined epithelial proliferation of more than two to three cell layers, with variable degrees of cytoplasmic and nuclear pleiomorphism. It may occur spontaneously, in association with chronic progressive nephropathy, or as a result of chemical administration. Atypical tubule hyperplasia usually arises from the proximal convoluted tubule, but a distal or collecting duct origin is also possible. It is considered a putative preneoplastic lesion that is part of the continuum, leading to neoplasia . In our Although only half of the elderly females in which renal tumour was recorded in the NTP study  showed sConcerning the gender differential in the OTA dose-response, the perception of a mechanism involving OTA binding to male-specific rat (and presumably mouse) serum alpha 2u-globulins and augmented delivery to nephron epithelia via glomerular filtration, as in alpha 2u-nephropathy, has been described . It coulThe relevance of some experimental whole rat data to humans may be uncertain as a model, but for OTA the rat has been the best available. Studies on putative molecular tumorigenic mechanisms have in recent years used the sophisticated methodologies that have become available. However, no clear relevant picture has emerged to explain how tumour occurrence is mainly unilateral with highly focal origin in just a few of the thousands of separate nephrons, and that requires the insults from the ingestion of the mycotoxin well into the µg/kg b.w. range during most of the first year of life. OTA has been administered experimentally in vitro to tissue-cultured cells or kidney homogenates without knowledge of the relevant kidney epithelial cell exposure to OTA during in vivo tumorigenesis, because that is unknown. Previously, an analysis of several London OTA/rat carcinomas did not identify evidence of mutations in the rat orthologues of human familial renal cell carcinoma genes VHL and FLCN , and genome-wide genetic sequencing studies are currently in progress. From the present immunohistochemical review of tumour heterogeneity a direct model for humans is uncertain, especially since a better affinity with spontaneous renal tumours arising in the Eker rat has recently been recognised also immunohistochemically . A comprTherefore, a provisional conclusion of the present pilot study is that the rat fails to provide any model support for an aetiological role for OTA in urothelial cancers sometimes associated with Balkan endemic nephropathy. Further, a predictive animal-model role for the rat in human RCC aetiology seems at present insecure. Notably in the particular case of the late Palle Krogh, whose premature demise from a metastasising RCC might have come from professional experimental exposure to OTA for several years, the primary renal tumour was diploid  in markeArchived paraffin-embedded tumorous kidneys from Fischer males E. The exThe 1989 NTP study  reports Where necessary, 3-µm sections were cut from paraffin blocks. Sections on charged slides were studied immunohistochemically, using both automatic and manual procedures in three different locations: Peterborough City Hospital, Peterborough, UK; St George’s Hospital, London, UK, and; County Hospital, Timisoara, Romania.The following antibodies were used: CK AE1/AE3, clone AE1/AE3 (Dako); CK MNF 116, clone MNF 116 (Dako); CK HMW, clone 34 βE12 ; CK7, clone OV-TL 12/30 (Dako); CK20, clone Ks 20.8 (Dako); p63, clone DAK-p63 (Dako); Vim, clone V9 ; CD10, clone 56C6 (Dako); Desmin, clone D33 (Dako); Smooth Muscle Actin, clone 1A4 (Dako); TTF-1, clone SPT24 (Novocastra), and; S100, polyclonal . The antibody titer varies depending on the commercial form used: fixed/prediluted , respectively, the optimal dilution of the concentrated form, individually adjusted according to the internal protocol of each laboratory. For S100, CD10, Vimentin and some cytokeratins (CKs), the immune reactions were repeated in all three locations by different methods with the same results. The rationale of using the above listed markers is explained in the discussion chapter under the renal cell origin section. Some of these antibodies have been applied selectively on a few cases only, where differential diagnosis was considered  for 10 min.Counterstain with Haematoxylin for 5 min.The staining protocol varied from site to site, but the main steps were:The brown immune reaction product of DAB chromogen is cytoplasmic and/or membranar for most of the listed antibodies, nuclear for p63 and TTF-1, and cytoplasmic and nuclear for S100. The immunoreactivity was evaluated by a percentage of positive tumour cells as diffuse for ≥50% and focal for <50%; the intensity of reaction was appreciated as weak or intense compared to control.


Knowledge of the intestinal stem cell niche gathered from both in vivo and novel in vitro models may help us improve therapies for tumorigenesis and intestinal damage and make autologous intestinal transplants a feasible clinical practice.Intestinal stem cells are located at the base of the crypts and are surrounded by a complex structure called niche. This environment is composed mainly of epithelial cells and stroma which provides signals that govern cell maintenance, proliferation, and differentiation. Understanding how the niche regulates stem cell fate by controlling developmental signaling pathways will help us to define how stem cells choose between self-renewal and differentiation and how they maintain their undifferentiated state. Tractable  The expression of the RNA-binding protein Mex3a labels a slowly cycling subpopulation of Lgr5+ ISCs that contribute to all intestinal lineages. Thus, Mex3a defines a reserve-like ISC population within the Lgr5+ compartment [in vivo and in vitro models for intestinal CBC stem cell niche.The intestine represents the most vigorously renewing adult tissue, which undergoes rapid turnover in order to prevent damage from stress factors; its tissue-specific stem cells are essential for tissue homeostasis in the adult organism . These upartment . Some repartment , 9–13. Fβ, and Hedgehog signaling pathways to maintain proliferation/differentiation balance [Control of proliferation, self-renewal, and lineage specification of the stem cells in the crypt are believed to be directed by an actively regulated process based on cell-cell and cell-stroma interactions . The ISC balance –19.in vitro systems allowing long-term culture and until some years ago, the only possible strategy to analyse such interactions for a potential role in intestinal development, homeostasis, damage or tumorigenesis was the time-consuming tissue-specific mouse models. For example, Achaete-scute complex homolog 2 (Ascl2) was reported to be responsible for controlling intestinal stem cell fate by using transgenic mice [+ stem cells are sufficient to give rise to organoids in culture which contain all differentiated lineages: Paneth cells at the base of the crypt and enteroendocrine, goblet cells, and enterocytes that migrate upwards the villus. Importantly, these cultures allow ex vivo monitoring intestinal stem cell function with respect to self-renewal and production of rapidly dividing crypt progenitor cells and differentiated lineages and are therefore comparable to the in vivo situation [in vivo models to the most novel in vitro technology which will improve in the next years our understanding of stem cell behavior.Functional analysis of stem cells and their environment has been hampered by a lack of suitable nic mice . In 2009nic mice –23, and nic mice , 25. Theituation . In thisβ-catenin, Tcf4 knockout models) or ectopic expression of the secreted Wnt antagonist Dickkopf-1 (Dkk1) disrupts intestinal epithelial homeostasis, leading to crypt loss, reduced proliferation, and altered differentiation [Adenomatous polyposis coli (Apc), drives hyperplasia and increases the expansion of intestinal stem cell niche [EphB2, EphB3, and their ligands ephrins, are key coordinators of migration and proliferation in the stem cell niche. EphB knockouts show that these proteins determine cell positioning along the crypt-villus axis in the intestinal epithelium [Stem cells require the support of neighbouring epithelial cells to maintain their function. The epithelial niche modulates several signaling cascades, the Wnt pathway being one of the main regulators of stem cell self-renewal. Genetic deletion of the Wnt pathway's main players , and EGF factors that regulate stem cell maintenance [−/− mouse model, transgenic expression of diphtheria toxin A under the Paneth cell-specific cryptdin 2 promoter (CR2-tox176), and conditional deletion of Sox9 showed that the stem cells were coincidently decreased in number [+ stem cells that contained a distinct cKit/CD117+ crypt base subpopulation which expressed the Notch ligands Delta-like (Dll) 1, Dll4, and EGF. In vivo, this colonic cKit population was regulated by Notch signaling [+-expressing deep crypt secretory (DCS) cells  that are intermingled with the Lgr5+ colonic stem cells at the base of the crypt. These cells also produce Wnt and Notch factors to support essential growth and maintenance signals. In these mouse experiments, the ablation of these types of cells resulted in loss of stem cell function and disruption of colon homeostasis [+ cells, the signals provided by them markedly increased differentiation and organoid growth from a single stem cell in the in vitro culture [“What is the main source of epithelial Wnt signals within the intestine?” The secretory Paneth cells adjacent to CBCs secrete Wnt3, and they constitute an important part of the small intestinal stem cell niche. They are known to produce bactericidal products such as lysozyme and cryptdins/defensins, and in addition, they can also produce TGFntenance , 35. Redn number . Some stn number . Althougignaling . Later oeostasis . When Pa culture , 38. Nov culture .+ stem cells are critically dependent on Notch, which depend on direct cell-cell contact as the Paneth cells are the main sources of Notch signals [+ SCs [Several Notch mouse models evidence the impact of this pathway in epithelial stem cell niche. Indeed, Lgr5 signals –46. Thiss [+ SCs . A negats [+ SCs .Leucine-rich repeats and immunoglobulin-like domains (Lrig) 1, a direct Myc target gene as part of a negative feedback loop that modulates the proliferation of intestinal progenitor cells. Lrig knockout mouse induces upregulation of EGFR, ErbB2, and ErbB3 promoting downstream activation of c-Myc within intestinal stem and progenitor cells [Other studies show or cells , 47. Theor cells . These aVigorous crosstalk between the epithelium and the underlying nonepithelial niche is required to define the crypt-villus axis. It is well established that mesenchymal cells secrete BMP antagonists such as Gremlin 1 and Gremlin 2 at the bottom of the crypts which supports compartmentalization . Thus, BMesenchymal cells also secrete Wnt proteins, and R-spondins have been detected in the intestinal stroma , 53–55. Angptl2 knockout mice reduces CBCs and influences Wnt pathway; however, ANGPTL2 is dispensable for intestinal homeostasis [All these data suggest that Wnt signals from Paneth cells can be replaced by stromal ones, so nonepithelial activation of Wnt pathway may support intestinal stem cell maintenance. On this direction, recent studies show that a subpopulation of mesenchymal cells marked by the winged-helix transcription factor Foxl1 is critical in maintaining stem cells. These cells produce Wnt factors, and their ablation reduces crypt growth. However, there is a need to better identify this subset of mesenchymal cells . In addieostasis   also influence the stem cell behaviour by modulating the different signaling cascades. They secrete growth factors, cytokines, and ligands that alter the stem cell fate , 59. UpcIn vitro models of the ISC niche circumvent these problems by offering a level of accessibility and tractability that is difficult or impossible to achieve in vivo.Animal models have provided invaluable insight into the nature and hallmarks of the intestinal stem cell, as well as the set of microenvironmental inputs that govern its behavior and constitute the intestinal stem cell (ISC) niche. However, in addition to ethical and practical considerations, animal-based studies suffer multiple limitations in the scope of scientific questions they can address. In particular, mouse models generally do not afford the dynamic and multifactorial observation and control that are required for securing comprehensive understanding of ISCs and their niche. Further, whereas the mouse intestine is an adequate approximation of its human counterpart, several crucial developmental and histological differences exist , and mouin vitro for several decades. A decade ago, Sasai demonstrated that, aside from directing pluripotent stem cells to commit toward a certain lineage, thus obtaining populations of differentiated cells, stem cells and their progeny can follow their innate developmental programs and self-organize into structures that mimic multiple histological and functional aspects of real organs [in vitro were termed organoids.Driven by both basic research and therapeutic objectives, researchers have cultured stem cells l organs , 62. Thein vivo, thus forming a bud structure of similar in shape and size to those of the intestinal crypt. Small modifications of the culture protocol—notably, the addition of Wnt3a—allow for the culture of adult human ISC-derived intestinal organoids [Intestinal organoids, or “miniguts”, generated in the laboratory of Hans Clevers, were among the first types of stem cell-derived organoids reported , 24. SatIn addition to adult ISCs, intestinal organoids have been generated from induced pluripotent stem cells (PSCs), using a protocol inspired by human embryonic development . PSCs wein vivo studies in our quest to dissect the ISC niche and define the mechanisms whereby it exerts its influence on stem cells. Indeed, to demonstrate that Paneth cells constitute the ISC niche, as discussed above, mouse models were used in conjunction with intestinal organoids [Aside from promising to revolutionize basic and clinical research, by serving as models of development and disease, platforms for drug discovery and toxicity screens and sources of tissue for cell-based therapies , 65, intrganoids , 66. Likrganoids , 68 and rganoids , 70 in tThe format of common organoid culture models allows for relatively easy and routine manipulations of the soluble microenvironment of ISCs. Beyond a set of soluble cues, the stem cell niche also comprises adhesion and mechanical signals from the surrounding ECM , which aSynthetic hydrogels, comprising a water-swelled polymer network, can be rendered biocompatible and biofunctional through the incorporation of essential biological signals and used as well-defined alternatives to animal-derived ECM gels, such as collagen and Matrigel –80. Morein vivo [in vivo. Future studies not only could take a system-level approach and expanded the number of ECM factors tested but also investigate potential interactive effects of multiple components. Sophisticated high-throughput approaches to generate and analyze multifactorial environments, which have already been used to study other stem cell systems [We recently took advantage of poly(ethylene glycol) (PEG) hydrogels to identify ECM components that control ISC fate and used this knowledge to construct well-defined and tunable matrices for the culture of ISCs and intestinal organoids  Figure . Inspirein vivo , 82–85,  systems , 87, seein vivo experience physical signals from the microenvironment, including the mechanical properties of their surrounding ECM. The mechanical environment is now recognized as a major extrinsic regulator of multiple stem cell systems [in vivo studies provide clues that mechanical forces may directly control ISC proliferation in the colon [In addition to soluble and tethered molecular factors, ISCs  systems . Our undhe colon . We usedhe colon  and obsehe colon , 91, andhe colon , 70, 81.in vivo and current organoid models is not only the types of niche components available but also the mode in which they are presented. Current organoid cultures contain essential niche signals, including Wnt and Notch pathway agonists, and BMP inhibitors. However, these soluble components are included in the cell culture medium, wherein they eventually reach a uniform concentration. In contrast, master regulators of intestinal biology in vivo are presented in distinct spatiotemporal patterns, which are crucial for the regionalization of the intestine and the establishment of the ISC niche. For example, Wnt signals in vivo are produced by the subepithelial mesenchyme and Paneth cell and thus restricted to the bottom of the crypts where they are crucial for maintaining ISCs in a self-renewing state [A key difference between the intestinal niche ng state . Bone mong state . This diin vitro model of a stable, homeostatic ISC niche (Bioengineers have developed a number of strategies for controlling the spatiotemporal patterns of soluble and tethered cues in soft 3D media, similar to the matrices required for organoid formation. Microfluidically generated morphogen gradients are perhaps the most widespread approach. Here, 3D hydrogels are microstructured using lithographic –95 or abSC niche .


Herein our aim was to determine the effect that reducing EnNaC activity with a very-low-dose of amiloride would have on decreasing endothelial and arterial stiffness in young female mice consuming a WD. To this end, we fed female mice either a WD or control diet and treated them with or without a very-low-dose of the ENaC-inhibitor amiloride (1 mg/kg/day) in the drinking water for 20 weeks beginning at 4 weeks of age. Mice consuming a WD were heavier and had greater percent body fat, proteinuria, and aortic stiffness as assessed by pulse-wave velocity than those fed control diet. Treatment with amiloride did not affect body weight, body composition, blood pressure, urinary sodium excretion, or insulin sensitivity, but significantly reduced the development of endothelial and aortic stiffness, aortic fibrosis, aortic oxidative stress, and mesenteric resistance artery EnNaC abundance and proteinuria in WD-fed mice. Amiloride also improved endothelial-dependent vasodilatory responses in the resistance arteries of WD-fed mice. These results indicate that a very-low-dose of amiloride, not affecting blood pressure, is sufficient to improve endothelial function and reduce aortic stiffness in female mice fed a WD, and suggest that EnNaC-inhibition may be sufficient to ameliorate the pathological vascular stiffening effects of WD-induced obesity in females.Obese premenopausal women lose their sex related cardiovascular disease protection and develop greater arterial stiffening than age matched men. In female mice, we have shown that consumption of a Western diet (WD), high in fat and refined sugars, is associated with endothelial dysfunction and vascular stiffening, which occur via activation of mineralocorticoid receptors and associated increases in epithelial Na Stiffening of the arterial vasculature is highly predictive of increased cardiovascular disease (CVD) including macro and microvascular associated pathologies such as coronary artery disease, peripheral artery disease, and stroke, as well as the development of proteinuria and chronic kidney disease   bioavailability, as well as with reductions in arterial fibrosis, oxidative stress, vascular wall hypertrophy, inflammation, presence of epithelial Na+ channels (ENaC) in the endothelium (EnNaC), endothelial stiffness and endothelial dysfunction. Our results, therefore, suggest that WD feeding induces arterial stiffening through a pathway that requires ECMR signaling and the subsequent augmented activity of EnNaC, endothelial stiffening, oxidative stress, and reduced NO bioavailability.Human population studies show that women with obesity and diabetes have a greater risk for CVD than comparable men  in response to insulin or shear stress , while ECMRBody composition was assessed using an EchoMRI-500 Analyzer  in 20-week old conscious mice as previously described  or vehicle control for 4 h before the AFM nano-indentation protocol was performed.The elastic modulus or stiffness of the aortic endothelial surface was measured in thoracic aorta explants using an atomic force microscope (AFM) MFP-3D AFM 89  as previously described  reduction of aortic medial thickness in animals treated with amiloride . In contrast, very-low-dose amiloride prevented the increase in proteinuria associated with consumption of a WD. There is increasing evidence which suggests that vascular stiffness predicts CKD and, in this context, measures of proteinuria are a marker of kidney function closely linked to vascular stiffness (Liu et al., In this investigation, we initially examined the effects of very-low-dose amiloride on body composition, glucose metabolism, blood pressure, renal Na+ ions. We found that the endothelial specific presence of ENaC was unaltered by amiloride in the aortae of female mice fed a WD. Therefore, it is likely that amiloride decreased EnNaC activity in the aorta without altering its expression. In this regard, 20 weeks of treatment with very-low-dose amiloride may have been insufficient to induce changes in aortic protein expression, or it is possible that this extended treatment period may have resulted in a compensatory upregulation of EnNaC in this conduit artery. Comparatively, we found that EnNaC presence was reduced in mesenteric arteries from WD-fed mice treated with amiloride. This finding is in line with previous data showing that acute treatment with amiloride reduced EnNaC expression in human umbilical vein endothelial cells (Kusche-Vihrog et al., ex vivo exposure of arteries explanted from human subjects to amiloride softened their endothelial surface (Lenders et al., ex vivo exposure to amiloride did not occur when subjects had significantly increased PWVs (Lenders et al., ex vivo. Results indicate that amiloride acting directly on vascular tissues is able to reduce endothelial stiffness, which further suggests that our in vivo treatment with amiloride likely reduced aortic stiffness in females consuming a WD by acting directly on EnNaC activity.As amiloride did not affect body composition, blood pressure, or insulin sensitivity in female mice fed a WD, our major finding that amiloride reduced aortic PWV in those mice suggests amiloride reduced vascular stiffness via direct actions on the vascular wall. It has been shown that the expression and activity of EnNaC increases cellular stiffness and reduces the capacity of endothelial cells to produce NO (Oberleithner et al., + entry into endothelial cells in response to increased reactive oxygen species and EnNaC activity should increase the stiffness of the endothelium and reduce its capacity to produce NO (Oberleithner et al., As mentioned above, increases in EnNaC activity reduce the capacity of vascular endothelium to produce NO (Oberleithner et al., In mesenteric arteries, the increased endothelium-dependent vasodilatory capacity induced by amiloride was associated with a reduced presence of EnNaC, but not with a reduction in vascular stiffness. Contrary to expectations, amiloride reduced the distensibility of mesenteric arteries obtained from female mice fed a WD. However, this reduced distensibility was associated with a diminished capacity of arteries to have smaller diameters at the lowest pressure tested, and not with a reduced maximal passive diameter at high pressures. This, in conjunction with the significant increase in F-actin, elastin, and collagen content in the wall of these mesenteric arteries, suggest that amiloride through its capacity to increase NO bioavailability favored the arterialization of these resistance vessels. Indeed, it has been previously shown that NO promotes the differentiation and maintenance of a vascular smooth muscle cell contractile phenotype with an increase in F-actin content and the overall arterialization of blood vessels, which specifically includes augmented extracellular matrix expression by differentiated vascular smooth muscle cells (Lincoln et al., ex vivo to amiloride (Jia et al., −/− female mice fed a WD to amiloride ex vivo, and found that exposure to amiloride did not improve flow-induced vasodilation (Figure + through EnNaC increases cytoskeletal actin fibers (Mazzochi et al., The major findings reported herein support our overarching hypothesis that aortic stiffening in females consuming a WD depends on activation of MRs in endothelial cells that in turn enhance the presence and activity of EnNaC to promote endothelial stiffness and dysfunction. Consequently, our specific hypothesis tested here was that reducing the activity of EnNaC with very-low-dose amiloride would decrease arterial stiffness in female mice fed a WD, and that this arterial de-stiffening would be accompanied by amelioration of arterial fibrosis, endothelial dysfunction, oxidative stress, and endothelial stiffening, which our results substantiate. Previously, we showed that isolated mesenteric arteries from wild type female mice fed a WD, which have increased vascular MR expression (Jia et al., n Figure . This fun Figure , which in Figure . ENaC isThis study was carried out in accordance with the recommendations of the guidelines from the National Institutes of Health. The protocol was approved by the animal care and use committees at the University of Missouri and the Harry S. Truman Veterans' Memorial Hospital.LM and JS conceived and designed the study, interpreted data, and edited the manuscript. AA, FR, GJ, JH, VD, and BB performed experiments, made figures, and analyzed data. LM, AW, and RN interpreted data and wrote and edited the manuscript. All authors approved the final version of the manuscript.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.