Shc protein and protein kinase C (PKC) in these pathologies by their intracellular regulation of redox balance and oxidative stress levels. Additionally, we discuss possible therapeutic strategies aimed at attenuating the oxidative damage in these diseases.Reactive oxygen species (ROS) are a byproduct of the normal metabolism of oxygen and have important roles in cell signalling and homeostasis. An imbalance between ROS production and the cellular antioxidant defence system leads to oxidative stress. Environmental factors and genetic interactions play key roles in oxidative stress mediated pathologies. In this paper, we focus on cardiovascular diseases and obesity, disorders strongly related to each other; in which oxidative stress plays a fundamental role. We provide evidence of the key role played by p66 Obesity, high blood pressure, insulin resistance, and aging are associated with the development of cardiovascular diseases (CVDs), and all these factors are correlated with metabolic syndrome (MS) . LifestyShc) and some isoforms of the protein kinase C (PKC) family that are particularly sensitive to redox stress and are implicated both in CVDs and obesity [This paper aims to illustrate the correlation between oxidative stress, obesity, and CVDs, especially focusing on the 66-kilodalton (kDa) isoform of the growth factor adapter Shc (p66 obesity .2 molecules (1–3%) taken up by cells are converted into superoxide anion radicals (O2•−) by complexes I and III. This radical can subsequently be diverted into hydrogen peroxide (H2O2) and the hydroxyl radical (OH•). Such oxidative species are considered normal metabolic by-products. They are continuously generated by mitochondria and are kept in check by endogenous cellular antioxidant mechanisms, such as superoxide dismutase (which rapidly converts superoxide into H2O2 and O2), catalase, glutathione peroxidase, and peroxiredoxins distributed throughout the cell.ROS generation can be finely controlled and can constitute a physiologic signalling pathway. The enzyme systems responsible for ROS generation, as well as the antioxidant defences, have specific subcellular localization and thus give rise to the concept of compartmentalization of both ROS production and the signalling response. ROS can originate from different subcellular sources, but mitochondria are generally considered the primary source of ROS generation , 10. ROSOxidative stress represents an imbalance between ROS production and the cellular antioxidant defence system. In stress conditions, ROS levels increase and, because of their high reactivity, participate in a variety of chemical reactions. They are involved in cell damage, necrosis, and apoptosis via oxidation of lipids, proteins, and DNA  and prov2•− mediates its effects within a short range of its production, H2O2 is more stable and can diffuse throughout the cell; hence, despite the compartmentalization of ROS production, electrons generated by excess mitochondrial metabolism can be used to regulate intracellular signalling through the production of ROS [2O2 can diffuse through aquaporin channels in the plasma membrane to elicit an intracellular signalling response. Aquaporins belong to a large family of proteins that form pores in the membrane and conduct water in and out the cell [2O2 has almost the same size, dielelectric properties, and capacity to form hydrogen bonds as does water. Bienert et al. provided molecular genetic evidence that aquaporins, and in particular hAQP8, AtTIP1;1, and AtTIP1;2, can channel H2O2 [2•− can initiate intracellular signalling by penetration of the cell membrane through anion channels  [ROS production can rise when the breakdown of metabolites in the tricarboxylic acid (TCA) cycle exceeds the capacity of the electron transport chain (ETC) to assimilate the resulting electrons . While On of ROS . Moreovethe cell , and H2Onel H2O2 . Also ex, ClC-3) .CVDs are a class of pathologies involving the heart or blood vessels . They refer to any disease that affects the cardiovascular system, mainly cardiac diseases, vascular diseases of the brain and kidney, and peripheral arterial disease. World Health Organization (WHO) data published in September 2012 define CVDs as the principal cause of death globally: more people die annually from CVDs than from any other cause.Oxidative stress has a central role in the pathogenesis of atherosclerosis; indeed, it is a critical feature in atherogenesis. An increased generation of ROS in the vascular wall and a reduction of nitric oxide (NO) bioavailability lead to endothelial dysfunction in atherogenesis , 18. ROS2•−. Important roles have been shown for NADPH oxidases in redox signalling events involved in hypertension, atherosclerosis, endothelial activation, and angiogenesis, as well as in endothelial dysfunction [2•− inactivates NO promoting endothelial activation [−/−) mice maintained on a high-fat diet, Nox2 deletion was associated with decreased aortic ROS production and markedly less atherosclerotic plaque formation [in vivo comes from experiments using transgenic mice to alter expression of mitochondrial antioxidant proteins. Initial experiments using genetic knockouts showed that mice lacking MnSOD produce huge amounts of mitochondrial ROS and develop cardiomyopathy within the first weeks of birth [An important source of ROS is represented by NADPH oxidases (Nox), a family of enzyme complexes that catalyze the transfer of electrons from NADPH to molecular oxygen to generate Ofunction . The clofunction . The excfunction , and O2tivation . Nox2 antivation . Interesormation . Recent ormation . Moreoveormation . Some ofof birth . NowadaySmoking, hypertension, and diabetes mellitus, which represent the main risk factors for atherosclerosis, are associated with an increased production of ROS by the endothelium . SmokingShc in ROS production within mitochondria and its involvement in CVDs [Shc is also present in mitochondria-associated membranes (MAMs) and its levels change in an age-dependent manner [Mitochondria are an essential ROS producer in heart and cardiovascular diseases. Several studies reveal the role of p66 in CVDs , 37. p66t manner , 39.Shc is a protein encoded by the ShcA gene [Shc has an additional collagen homologous region (CH2) at its N-terminus [p66hcA gene , 41 thatterminus . Shc is very important for the regulation of the intracellular redox balance and oxidative stress levels. Many studies now support the fact that intracellular free radicals are reduced in cells lacking the p66Shc gene. There are three mechanisms that involve p66Shc in ROS formation. In the nucleus, p66Shc inhibits the FOXO transcription factors, causing a decrease in the expression of ROS scavenging enzymes [Shc promotes rac1 activation and triggers NADPH membrane oxidase ROS production. In addition, p66Shc acts also in the mitochondrial intermembrane space (IMS). After serine phosphorylation by PKCβ and prolyl-isomerization by Pin-1 [Shc moves from the cytosol to the IMS, through the TIM/TOM mitochondrial import machinery. Here, a redox active region at its N-terminal mediates electron transfer from reduced cytochrome c to molecular oxygen and the production of H2O2 [β, causing phosphorylation of p66Shc and thus triggering its mitochondrial proapoptotic effects [Shc induces mitochondrial H2O2 production and so further increases intracellular H2O2 levels; therefore, in this way it can maintain or increase PKCβ activation in a kind of self-triggered control loop  mice are protected against vascular, cardiac, and renal impairment. On the contrary, overexpression of p66Shc causes alteration of the mitochondrial network, leading to cytochrome c release and apoptosis. Napoli et al. demonstrated that mice with comparable lipid profiles, both in a low-fat condition as well as in a high-fat diet, had an increased early aortic lesion in p66Shc wild-type strain, whereas p66Shc−/− were protected. Of relevance, low predisposition to atherogenesis and reduced oxidative stress were coupled with reduced apoptosis in aortic lesions [The known role of p66 lesions . −). To demonstrate p66Shc involvement, Menini et al. and Rota et al. carried out several studies on hyperglycaemia-induced ROS-mediated cardiovascular complications, and p66Shc−/− mice were protected from cardiomyopathy [Shc−/− diabetic mice showed an enhanced antioxidant defence and lower ROS generation [Shc is involved in endothelial dysfunction, vascular dysfunction and plaque formation [Shc−/− mice display decreased ROS production and decreased myocardial injury caused by postischemic reperfusion [Shc gene expression levels in peripheral blood monocytes (PBMs) were significantly higher in coronary artery disease (CAD) patients, compared to non-CAD subjects [ROS generation is also one of the main pathophysiological mechanisms that links glucose metabolism to endothelial dysfunction and atherosclerosis. Hyperglycaemia plays also a central role in causing diabetic vascular complications. In particular, high glucose concentrations induce cellular events that increase the production of free radicals, which scavenge NO to form peroxynitrite  family. ROS trigger PKC through redox signalling: oxidation of critical cysteine residues on PKC isoforms is known to cause their activation and thus provides a mechanism by which ROS could turn on PKC.ε confers cardiac protection, whereas its selective inhibition abolishes protection induced by IPC [ε activation and its translocation into mitochondria where it mediates several cardioprotective-signalling pathways and promotes cell survival [δ causes increased damage from ischemic insults both in neonatal cardiac myocytes and in adult isolated rat cardiac myocytes, whereas its inhibition results in protection [δ activation and leads its translocation to mitochondria and induction of cell death [δ, the generation of ROS is in turn controlled by PKCδ. Knockout mice lacking PKCδ exhibit a loss of ROS formation by the endothelium when subjected to cell stress agents such as UV and TNF-α and are resistant to death induced by H2O2 [Several works have identified critical roles for PKC family members in programming aspects of heart failure pathogenesis. Their activation can be cardioprotective and may mediate ischemic-preconditioning-(IPC-) induced protection . Selectid by IPC . During survival . In contotection . The masll death . In addi by H2O2 .2•− production, and both PKC and NADPH oxidase inhibitors are able to block this effect [PKCs are also involved in the activation of NADPH oxidase, a source of oxidative stress in vascular tissue of diabetes and insulin resistance state. Angiotensin II (ATII) has also been reported to induce Os effect . Experims effect , 63. Obesity is a metabolic disease with pandemic proportions, against which no effective pharmacological treatments have been found so far. Obesity is defined as an excess accumulation of adipose tissue. During obesity, the excessive accumulation of lipids overstimulates the adipose tissue development by an increase in preadipocyte proliferation, differentiation into adipocyte, and size of mature adipocytes . ObesityIt has been reported that obesity may induce systemic oxidative stress. Biomarkers of oxidative damage are higher in individuals with obesity and correlate directly with Body Mass Index (BMI) and the percentage of body fat ; in contHyperglycaemic conditions and oxidative stress accelerate also the generation of advanced glycation end-products (AGEs), a complex group of compounds that derives from reaction between reducing sugars and amino residues present in proteins, lipids, and nucleic acids , mediatiob/ob mice were reported to show upregulation of ER stress markers such as BiP, phosphorylated PERK, and phosphorylated α-subunit of eukaryotic translational initiating factor 2 (eIF2α) in adipose tissue and the liver [Both mitochondrial and endoplasmic reticulum (ER) stress responses can regulate or induce adaptation to the ROS production initiated by nutrient excess. Recently, he liver . Intereshe liver . Howeverhe liver .In recent years, novel roles have been assigned to ROS, notably their involvement in the control of body weight by the central nervous system. Specifically, the location where ROS exert these newly described roles is the hypothalamus, where numerous neurons control our satiety, while others control our hunger behaviour. Such roles have been implicated as contributing factors underlying diverse findings such as the age-related decrease ability to lose weight and the caloric restriction-induced longevity .A final important point to take into account is that epidemiological evidence clearly indicates that overnutrition at an organismal level also contributes to cancer development, so obesity is also associated with increased risk for several types of cancer , 84. Theβ activation [β induces p66Shc phosphorylation, thus allowing p66Shc to be recognized by Pin1, isomerized and imported into mitochondria, where p66Shc acts as ROS producer and so further increases intracellular ROS levels  and insulin resistance, which could contribute to obesity-associated diabetes and CVDs [in vivo. Therefore, oxidative stress is induced by obesity, but at the same time it promotes fat accumulation. Lee et al. demonstrated that H2O2-induced oxidative stress facilitates the differentiation of preadipocytes into adipocytes by accelerating mitotic clonal expansion. This effect was explained through the positive regulation of major transcriptional activators such as CCAAT/Enhancer Binding Protein-β (C/EBP-β) and Peroxisomal Proliferator Activated Receptor-γ (PPAR-γ), which are able to coordinate the expression of genes involved in the adipocyte differentiation program [β and PPAR-γ expression, as well as adipogenic differentiation in 3T3-L1 preadipocytes [Data throughout the literature indicate that an increase in ROS significantly affects white adipose tissue biology and leads to deregulated expression of inflammatory cytokines such as Tumor Necrosis Factor-and CVDs . Moreove program . Antioxiipocytes , 91. NACipocytes . Finallyipocytes .β [βI blocked adipogenesis, suggesting that PKCβI is required in the induction of adipogenesis in 3T3-L1 preadipocytes and adipocytes. Subsequent studies demonstrated that mice lacking PKCβ showed decreased fat in adipose tissue, liver, and muscle. These mice consumed 20–30% more food than wildtype, yet lost body weight, and the size of white fat depots was markedly decreased compared with that of wild-type litter-mates. The protection from obesity involves elevated oxygen consumption/energy expenditure and increased fatty acid oxidation in adipose tissue with concurrent increased mitochondrial biogenesis, upregulation of PGC-1α and UCP-2, and downregulation of perilipin [β are resistant to HFD-induced obesity, showing significantly reduced white adipose tissue (WAT) [β expression in obesity-prone C57BL/6J mice, specifically in WAT. Basal PKCβ expression was also found to be elevated in WAT of obese ob/ob mice. Remarkably, PKCβ−/− mice exhibited changes in lipid metabolism gene expression, and such alterations were accompanied by significant changes in serum adipokines [Aguiari et al. attributed an important role in adipogenic differentiation of mesenchymal stem cells, from both adipose tissue (adipose-derived stem cells (ADSc)) and muscle (muscle-derived stem cells (MDSCs)), to ROS and downstream effector kinases, in particular PKCβ . The serβ , 94, 95.β  showed terilipin . Moreoveue (WAT) . HFD selipokines .β may lead to loss of body fat and may suggest novel therapeutic strategies for obesity and obesity-related disorders. In support of this notion, PKCβ antagonists are currently undergoing clinical trials to reduce diabetes-linked complications [β activation, as revealed both by the strong inhibitory effect of a specific PKCβ inhibitor (hispidin) and through its genetic downregulation using siRNA [β inhibitor, along with APDs, could prevent or delay the development of obesity and obesity-related disorders, introducing the hypothesis that the inhibition of PKCβ could be therapeutically useful in conjunction with APDs  triggered by insulin and nutrients in leptin-deficient LepOb/Ob mice, a genetic model of obesity and IR. p66Shc promotes the signal inhibitory phosphorylation of insulin receptor substrate 1 (IRS1) by connecting it with mTOR effector S6 kinase, demonstrating p66Shc as a mediator of IR by excess nutrients [Shc−/− mice have confirmed the role of p66Shc in insulin signalling; they have demonstrated that it is also the overexpression of fat of another isoform of Shc locus, p46, that is the likely cause of decreased adiposity and reduced insulin sensitivity [ROS are also critical determinants of aging and age-associated diseases. PKC protein . Berniakelopment . Moreove to cold , 102. Fufunction  may be autrients . Recent sitivity .β-carotene and lycopene, that are fat-soluble and can function as free radical scavengers to decrease initiation and propagation of fatty acid oxidation [Antioxidants are agents that at low concentrations prevent or inhibit oxidation of oxidisable biomolecules, such as DNA, lipids, and proteins . Superoxxidation .Antioxidants have been tested in several experimental and clinical models with mixed success. Lane et al. conducted a population-based study to examine the association between consumption of certain nutrients and prevalence of peripheral arterial disease (PAD), and they found that increased consumption of antioxidants, vitamin E and C was associated with reduced odds of PAD . Other sTherefore, regardless of these controversial data from clinical studies with no significant effects for the set up of appropriate treatments based on antioxidants, oxidative stress still remains a potential attractive target for CVDs prevention and therapy. Possible future therapies aimed at decreasing mitochondrial oxidative damage should also be considered.In obesity, targeting adipocyte mitochondrial ROS production and increasing the overall antioxidant defence system are a challenge. A recent study suggested that antioxidant polyphenols  can increase the antioxidant capacity of the body against obesity-induced oxidative stress through the prevention of mitochondrial alterations, while totally or partially protecting the cells against the consequences of oxidative stress .Therefore inclusion of antioxidants in the diet may be indicated; indeed many foods, such as vegetables, fruits, red wine, and olive oil, contain phytonutrients that are soluble and can increase the antioxidant capacity , 116.In this paper, we have focused on the involvement of oxidative stress in CVDs and obesity, in light of the fact that a strong correlation between these pathologies has been observed. Adipose tissue, particularly visceral fat, is in fact associated with the pathogenesis of diabetes, hypertension, and heart disease , 118.Shc, and some isoforms of PKC family are relevant participants in this mechanism.ROS play an important role through highly regulated redox-sensitive signalling pathways, the adaptor protein p66The use of antioxidants appears to be positive for the prevention of CVDs, while inhibitors of ER stress can represent novel drug targets for obesity. Understanding molecular links is fundamental to design new therapeutic strategies aimed at reducing the risk of developing these pathologies.


To determine whether in utero programming and immunomodulation also affect insulin resistance (IR) and type 2 diabetes, we investigated the effects of immunization on glucose and insulin responses in LDL receptor-deficient mice fed regular or 60% sucrose diets, as well as in offspring fed 0.5% cholesterol or 60% sucrose diets. IR was assessed by fasting glucose and insulin levels, oral glucose tolerance tests, glucose clamps, pancreatic immunohistochemistry and plasma free fatty acid concentrations. Immunizations improved glucose responses in both genders and protected both immunized mice and their offspring against IR and type 2 diabetes. Protection occurred even under euglycemic conditions, but was greatest in obese males exposed to very obesogenic/diabetogenic conditions. Hyperinsulinemic euglycemic clamps confirmed that maternal immunization protected mainly by reducing IR, but pancreatic immunocytochemistry also indicated some protection against beta cell damage. Maternal immunization was associated with marked regulation in offspring of 4 genes relevant to diabetes and 19 genes of importance for oxidative stress, as well as increased hepatic activities of key antioxidant enzymes. These findings establish that targeted immunomodulation may be used to protect immunized subjects and their offspring against IR and type 2 diabetes, and thus to reduce cardiovascular risk. They also support the notion that in utero programming influences offspring disease not by a single mechanism, but by multiple systemic effects.Maternal immunization with oxidized lipoproteins prior to pregnancy protects against atherogenic  The increasing prevalence of maternal obesity and related dysmetabolic conditions is therefore expected to lead to a wave of cardiovascular disease in their children, but little is known about the mechanisms of pathogenic in utero programming and its prevention. However, studies on one such maternal dysmetabolic condition, hypercholesterolemia, have shown that interventions in mothers may provide life-long benefits to offspring in utero had greater overall mortality than those who inherited FH from their father also supports the pathogenic role of maternal hypercholesterolemia Extensive epidemiological evidence links the in utero programming of IR Some of the mechanisms by which maternal hypercholesterolemia affects developmental programming have already been established, for example the increased oxidative stress associated with elevated cholesterol levels in utero programming is the possibility of achieving long-term protection of offspring by brief interventions in mothers. Cholesterol-lowering by cholestyramine or antioxidant treatment during pregnancy markedly reduces postnatal atherogenesis in rabbits and mice in utero immune programming would not only reduce atherosclerosis, but also the susceptibility to other conditions influenced by oxidative stress, immune mechanisms and inflammatory processes, including IR and type 2 diabetes.From a translational perspective, the most important aspect of in utero programming of IR and that immunization with oxidized lipoproteins protects immunized murine mothers and their offspring against IR and diabetes under euglycemic, hypercholesterolemic, and hyperglycemic conditions. For this purpose, we investigated the effects of immunization with LDL oxidized to various extent on glucose and insulin metabolism. This was done both in immunized subjects, because altered glucose metabolism in immunized mothers may influence in utero programming, and in offspring, to establish the effects of in utero programming on IR and type 2 diabetes. Given that moderate protective effects may be overwhelmed by harsh diabetogenic conditions, offspring were exposed to either a moderately obesogenic diet consisting of standard chow enriched with 0.5% cholesterol or an obesogenic diet containing 60% sucrose. Finally, effects on IR in males and females were investigated separately, because the antiatherogenic effects of maternal immunization are greater in male mice than in females We now provide evidence that maternal immunomodulation indeed affects −/−) mice from our colony, established from founders bred back into the C57BL/6 background for 10 generations. To determine the effects of immunization in immunized subjects and in offspring of both genders under different dietary conditions, seven experiments were carried out. Their purpose and experimental design is outlined in Experiment 1 investigated the effects of immunization with naturally oxidized LDL (nLDL) in 24 immunized females and 25 non-immunized controls. Immunization (primary and 2 boosts) began at age 6 weeks and oral glucose tolerance tests (OGTTs) were performed 40 or 250 days thereafter. Experiment #2 tested the effect of maternal nLDL immunization in male offspring of 24 nLDL-immunized and control mothers fed regular chow and bred after completion of immunization. From their offspring, two groups of male offspring roughly matched for number, birth dates and body weight were assembled . Upon weaning at 4 weeks, offspring were fed regular chow supplemented with 0.5% cholesterol , and OGTTs performed after 29 weeks. Experiment 3 investigated the direct effect of immunization with extensively oxidized LDL (OxLDL) under severely obesogenic and diabetogenic conditions in 22 males of the parental generation. In contrast to the first two experiments, this and the following experiments included an additional control group immunized with PBS. Immunized males, age 4 months, were fed a high-carbohydrate, low-fat diet termed 60% Sucrose  for 38 weeks. Experiments 4 and 5 investigated the effects of maternal OxLDL immunization on 65 male and 60 female offspring, respectively, exposed to severely diabetogenic conditions. In order to ensure that sufficiently large offspring groups matched for age, gender, and weight would be generated, 54 mothers fed regular chow were immunized with OxLDL or PBS, or served as untreated controls. Their male offspring were fed the 60% Sucrose diet for 28 or 38 weeks, beginning at weaning . Female offspring were fed the same diet for 24 or 36 weeks . Effects on glucose and insulin responses in Experiments 1–5 were assessed by OGTTs, hyperinsulinemic euglycemic clamps, pancreatic immunohistochemistry, qRT-PCR of mRNA expression in hepatic and cardiac tissues, and measurement of antioxidant enzyme activities. Differences in the length of dietary exposure in experiments 4 and 5 were mainly due to our logistical inability to perform all measurements at the same time, in particular clamps requiring surgeries and rate-limiting equipment. Experiment 6 compared the effects of the three experimental diets  in 31 males after 11 weeks. Experiment 7 compared free fatty acids in 32 offspring of OxLDL-immunized and control mothers fed 60% sucrose for 24 weeks, as well as 11 control mice fed regular chow .Experiments were carried out in 365 LDL receptor-deficient  Mice were immunized with homologous nLDL, OxLDL, or PBS. LDL was isolated by density gradient centrifugation Blood (20–25 µl) was collected from the retro-orbital plexus of 6 hour- fasted mice briefly anesthetized with isofluorane, using a precision vaporizer, prior to glucose gavage and 15, 30, 60, 90, and 120 min thereafter. Gavage consisted of intragastric administration of 1.5 g glucose/kg BW. Glucose was determined by Ascensia Contour glucometer and blood glucose test strips , and plasma insulin levels by Mercodia Ultrasensitive Mouse Insulin Kits .Mice were anesthetized with isofluorane  and a saline-filled 25-gauge catheter was implanted under sterile conditions in the right jugular vein, using a Leica MZ6 stereo microscope . Catheters were exteriorized through the dorsal neck. Incision sites were treated with lidocaine and sealed with surgical glue. Body temperature was maintained throughout surgery by a heated circulating water blanket. Animals were then kept on a swivel tether system  with free access to food and water for 48 hours, during which the catheter was perfused with 40 µl/hr saline with a PHD 2000 perfusor pump . After careful consideration of the various conditions of clamps in the literature The cholesterol content of lipoprotein fractions in the plasma was determined by fast-performance liquid chromatography (FPLC) Approximately 30 mg of liver tissue or 20 mg of cardiac ventricle tissue were dissected and placed into RNAlater . RNA was isolated using NucleoSpin mini RNA isolation kits  according to the standard protocol. Preparation of ventricular tissue included an extra proteinase K digestion step. RNA was quantitated using a spectrophotometer .www.primerdesign.eu). Selected probes were ACTB (beta actin cytoplasmic) and RPL13a .We first performed conventional qPCR to determine suitable normalization genes. For this purpose, 1 µg of total RNA was reverse transcribed, using the Clontech Sprint RT complete double primed kit (#PT4048-2) according to the protocol. The resulting cDNA was diluted 1∶40 (liver) or 1∶8 (ventricle). Quantitative PCR was performed with a Rotor-Gene 3000 PCR , using LUX Primers . Cycling conditions were: 1 cycle of 95°C for 2 min; 50 cycles of denaturation 95°C for 5 sec, hybridization/elongation 60°C for 20 sec, followed by a melt curve analysis ranging from 60°C to 95°C, ramp time 1°C per step, waiting for 60 sec for the first step and 5 sec for all subsequent steps. The PCR mix contained 1x JumpStart Taq Ready Mix  and 5 µl of diluted cDNA in a total volume of 25 µl per reaction. Normalization genes were selected from 12 candidates using GeNorm kit for mouse perfect probe (# ge PP-12-mouse) and qBase plus software (2 First Strand cDNA synthesis kit #330401 (Qiagen) containing RNA quality and genomic DNA contamination controls. qPCR was performed on a Roche Lightcycler 480 using the corresponding RT2 SYBR green Master Mix #330502 (Qiagen). cDNA synthesis and qPCR were performed according to manufacturer’s protocols. Array data were analyzed with the manufacturer’s online data analysis software , using the same normalizing genes we had previously chosen with GeNorm.Hepatic expression of 84 genes related to oxidative stress and 84 genes of interest for diabetes was then assessed with the Oxidative Stress and Antioxidant Defense qPCR Array (PAMM-065) and the Diabetes qPCR Array (PAMM-023), respectively (SA Biosciences/Qiagen). Reverse transcription was performed using 1 µg of total RNA and the RTApproximately 50 mg liver and 60 mg ventricle tissue were flash frozen in liquid nitrogen and stored at −80°C until processing. Tissues were placed in 500 µl ice cold PBS containing 1 mM EDTA, homogenized with a tissue homogenizer  and transferred into an Eppendorf tube containing another 500 µl ice cold PBS with 1 mM EDTA. The homogenate was centrifuged for 5 min at 1500×g at 4°C, and an aliquot of the supernatant was removed for measurement of total superoxide dismutase (SOD) activity. The remaining homogenate was then centrifuged at 10,000×g for 15 min and the supernatant was aliquoted for determination of cytosolic SOD, glutathione peroxidase (GPx), and catalase (CAT) activities. The pellet was resuspended in 300 µl of SOD sample buffer and again homogenized for mitochondrial SOD measurement. Samples from all mice were stored at −80°C for joint evaluation in the same assay. Activities were determined with Superoxide Dismutase, Catalase, and Glutathione Peroxidase Assay kits  according to the manufacturer’s protocols, and absorptions measured with a Synergy 2 Multi-Detection Microplate Reader . Protein concentrations were determined by BCA Protein Assay .The pancreas was carefully dissected under a Leica MZ6 stereo microscope , paraformaldehyde-fixed, and paraffin-embedded for immunohistochemistry. Sections (6 µm thick) were immunostained with 1∶1000 dilutions of mouse monoclonal antibodies against glucagon or insulin , using a Mouse-on-Mouse kit  and an avidin-biotin-alkaline phosphatase detection system with a red color substrate . Sections stained without primary antibodies served as controls. Glucagon-positive pancreatic alpha cells and insulin-positive beta cells were visually identified and counted. Results were expressed as cells per section or beta cell/alpha cell ratios.Normal-distributed data were compared by Student’s unpaired two-tailed T-test and differences considered significant when P≤0.05.-/- mice, but the 60% sucrose diet was more obesogenic and affected lipoprotein profiles and OGTT glucose responses to a greater extent, especially in males (A–D). The effect of this diet on plasma concentrations of 33 free fatty acids (FFAs) were assessed in males fed 60% sucrose or regular diet for 24 weeks. Representative FFAs with the highest concentrations are shown in (E–I). Most saturated and monounsaturated FFAs followed the patterns shown for 14∶0, 18∶0 and 16∶1, i.e. were greatly increased by the sucrose diet. Maternal OxLDL immunization tended to reduce this increase. This was statistically significant for some FFAs not shown, e.g. 20∶0 and 22∶1 (P<0.01 for both), but did not come close to compensating for the effect of the sucrose diet. In contrast, many polyunsaturated FFAs were decreased by the sucrose diet, again with only marginal influence of maternal immunization . As a result of these opposing trends, total FFAs were not significantly affected by the sucrose diet, and maternal immunization showed only a slight trend towards lowering them (J).The effects of the diets administered are compared in . All mice gained weight over time, but immunization had no effect on body weights (A). Better glucose responses in immunized subjects were first noted at day 40 (B), and became more evident 250 days after primary immunization (C). Insulin measurements showed a clear trend towards higher levels than in nLDL-immunized mice (D). The observation of higher glucose levels despite greater insulin release suggests that older control females develop some degree of IR even on regular chow, and that nLDL-immunization is protective even under conditions of euglycemia (and moderate spontaneous hypercholesterolemia).Immunization with either nLDL or OxLDL reduces atherogenesis in immunized animals . Time-averaged plasma cholesterol levels and terminal body weights were similar (data not shown). An OGTT performed at the end of dietary intervention showed better glucose responses in offspring of nLDL-immunized mothers (A). Fasting glucose was similar in both groups, but glucose at most time points after gavage and the area under the curve  was significantly lower in offspring of immunized mothers, compared to offspring of controls, even though OGTT insulin peaks were higher in the control group, consistent with greater compensatory hyperinsulinemic responses and greater IR in controls (B).To explore the effects of maternal immunization on offspring glucose and insulin metabolism under mildly obesogenic conditions, male offspring of nLDL-immunized mothers and non-immunized controls were fed the 0.5% cholesterol diet for 29 weeks, starting at weaning . Compared to non-immunized controls fed regular chow, all three groups on sucrose diet gained substantial weight (A). An OGTT at the end of the dietary intervention showed significantly better glucose responses in OxLDL-immunized males than in PBS-immunized and non-immunized controls, similar to those of normal non-obese mice fed regular chow . Fasting insulin and insulin responses after gavage were significantly lower in OxLDL-immunized mice than in both sucrose fed controls . Again, the higher glucose levels concomitant with greater compensatory insulin responses in the control groups are indicative of IR, and OxLDL immunization protected against the onset of IR induced by this extreme diet. Together, results of Having established that nLDL exerts beneficial effects on glucose and insulin responses in immunized females and their offspring under mildly obesogenic conditions, we then investigated whether OxLDL protects immunized mice under highly obesogenic conditions. For this and all subsequent experiments, we included an additional control group immunized with PBS (with FA). Based on previous atherosclerosis studies, we anticipated that this would yield intermediate results, due to the presence of oxidation-specific epitopes in the mycobacterial adjuvant and the generation of additional oxidative neoepitopes by the local inflammation caused by FA. We first tested the direct effect of OxLDL-immunization in older males immunized at age 4 months and fed the 60% sucrose diet for 30 weeks . After 28 weeks on 60% sucrose diet, no effect of immunization on body weights were noted, but offspring of OxLDL-immunized mothers showed significantly better glucose responses than controls (A). In contrast to directly immunized males ((B). To assess whether part of the protective effect may be due to greater protection of beta cells in offspring of OxLDL-immunized mothers, pancreatic immunohistochemistry was performed. No differences in the number of glucagon-positive alpha cells  or insulin-positive beta cells (F–H) per section were detected. However, the beta cell/alpha cell ratio showed a clear trend of being higher in offspring of OxLDL-immunized mothers than in offspring of PBS-immunized and control mothers . Thus, immunization may also convey some protection against beta cell deterioration.We then investigated the effects of maternal OxLDL immunization on IR in male offspring exposed to highly obesogenic conditions ed males , offspri(I) and fasting glucose levels (J) were similar in both groups, whereas the amount of glucose infused during the 30 min euglycemic period was markedly greater in offspring of OxLDL-immunized mothers (K). Maternal OxLDL-immunization therefore protects against IR, in addition to any protection it may convey against adverse effects of the sucrose diet to beta cells.To establish beyond doubt that OGTT results indicate a protective effect of maternal immunization against offspring IR, hyperinsulinemic euglycemic clamps were performed on two additional groups of male offspring of OxLDL-immunized and non-immunized mothers fed 60% sucrose for 38 weeks. Body weights , female offspring still weighed significantly less than males (A), but glucose (B) and insulin (C) responses resembled those in male offspring . After 24 weeks ffspring , establiadverse long-term effects of the diet, additional batches of female offspring were exposed to 36 weeks of sucrose diet followed by 19 weeks on regular chow. At age 59 weeks, body weighs (D) showed only a marginal increase, compared to 28 weeks (A), but fasting glucose levels were still significantly lower in offspring of OxLDL-immunized mothers than in offspring of controls (E). Furthermore, 7 of 17 animals in the control group were hyperglycemic (fasting glucose ≥230 mg/dl), compared to only 1 of 15 in the OxLDL group . Insulin responses also deviated from those previously observed in IR mice, in so far as controls failed to generate sufficient insulin to maintain euglycemia. Together, these results indicate that maternal immunization not only protects offspring against IR, but also delays the transition to type 2 diabetes.To follow the effects into old age without incurring in utero programming on hepatic gene expression and activity of antioxidant enzymes in the liver and cardiac ventricle of offspring. Both parameters were measured in male offspring after 28 weeks on sucrose diet characterized in .Given that oxidative stress is prominently involved in atherogenesis and, putatively, also in IR, we then investigated effects of , with a 35% and 32% increase in cytosolic and total SOD  , a 29% increase in GPx (P = 0.05) (C), and a trend towards an 11% increase in catalase (D). Mitochondrial SOD activity, too, was significantly greater in offspring of OxLDL immunized mothers (E). Higher hepatic glutathion (GSH) concentrations (F) and GSH/GSSG ratios (not shown) were also consistent with a better overall antioxidant protection of offspring of OxLDL-immunized mothers. Antioxidant enzyme activities in the ventricular part of the heart were much lower than in the liver and no differences were seen in the activities of cytosolic SOD, total SOD, catalase (data not shown) and mitochondrial SOD (G). GPx activity was actually lower in offspring of immunized mice (H). This suggests that in utero programming is cell- or organ-selective.Hepatic activities of key antioxidant enzymes were consistently higher in offspring of OxLDL-immunized mothers than in controls in utero programming by maternal hypercholesterolemia influences IR and diabetes. An influence of in utero conditions on diabetes has long been assumed, based on epidemiology, but the association was mainly with low birth weight and maternal factors impairing intrauterine growth in utero programming in our LDLR−/− mice most likely resulted from the same factors causing atherogenic programming in this and other animal models, i.e. hypercholesterolemia and associated oxidative stress The present study allows several conclusions. First, it shows that in utero programming of diabetic conditions can be inhibited by maternal immunomodulation, in analogy to the inhibition of atherogenic in utero programming. Notably, the protective effect was seen even in non-obese euglycemic females. To what extent the protective effect is due to the prevention of diabetogenic programming, as opposed to beneficial effects of immune programming resulting from maternal immunization in utero programming of a few individual genes or gene products.Second, and more importantly, the fact that maternal immunization prior to pregnancy reduces the susceptibility to IR and diabetes in offspring shows, in principle, that in utero programming affects diseases, the direct effects of OxLDL immunization and the mechanisms by which they may protect immunized subject or affect in utero programming are known. Oxidation-specific epitopes are a prominent feature of atherosclerotic lesions and are enhanced in diabetes by interactions between oxidation and nonenzymatic glycation (“glycoxylation”) in utero programming by oxidized fatty acids and other mediators of maternal oxidative stress capable of crossing the placental barrier In contrast to the mechanisms by which Third, the protective effect noted in both immunized animals and their offspring may provide new insights into the pathogenesis of IR and type 2 diabetes. The search for causes of IR has long focused on genetic defects in insulin receptors, insulin receptor ligands, and their downstream signaling, as well as on factors impairing insulin sensitivity that are typically increased in obesity, such as non-esterified fatty acids Our results raise the possibility that other maternal immunomodulation may also protect offspring against diabetic conditions. Clearly, offspring are not protected against the full spectrum of antigens the mother is immune to. In the case of maternal immunization with OxLDL, immune protection later in life may depend on natural post-natal boosting provided by the ubiquitous occurrence of the antigen and its increased formation by high-risk diets. However, there is no reason to believe that maternal immunization with diabetes-specific antigens may not also be protective, in particular if such immunity were deliberately enhanced after birth.in utero programming of aging processes Potential caveats: Differences in the genetic background of various murine strains are known to greatly influence their susceptibility to inflammation and obesogenic or atherogenic diets. It is therefore important that consistent findings were obtained under a broad range of conditions ranging from euglycemia in females exclusively fed regular chow to extensive IR in highly obese sucrose-fed males. Another caveat is the difficulty to separate effects on IR from those on aging. Antioxidant protection decreases with age, whereas obesity, IR and diabetes tend to increase. We cannot exclude that some of the protective effects are due to effects of immunization on From a clinical perspective, the most important insights are that IR can be attenuated by immunization and that maternal immunomodulation can reduce diabetic conditions in their offspring. Immunization of prospective mothers before pregnancy, ideally before they develop hypercholesterolemia or obesity, avoids the risks of pharmaceutical interventions during pregnancy, but may effectively protect offspring against IR and atherosclerosis. Results also emphasize the importance of preventing gestational hypercholesterolemia and obesity by conventional approaches and the persistent benefits for offspring of brief maternal treatment.


Aberrant DNA methylation is an important cancer hallmark, yet the dynamics of DNA methylation changes in human carcinogenesis remain largely unexplored. Moreover, the role of DNA methylation for prediction of clinical outcome is still uncertain and confined to specific cancers. Here we perform the most comprehensive study of DNA methylation changes throughout human carcinogenesis, analysing 27,578 CpGs in each of 1,475 samples, ranging from normal cells in advance of non-invasive neoplastic transformation to non-invasive and invasive cancers and metastatic tissue. We demonstrate that hypermethylation at stem cell PolyComb Group Target genes (PCGTs) occurs in cytologically normal cells three years in advance of the first morphological neoplastic changes, while hypomethylation occurs preferentially at CpGs which are heavily Methylated in Embryonic Stem Cells (MESCs) and increases significantly with cancer invasion in both the epithelial and stromal tumour compartments. In contrast to PCGT hypermethylation, MESC hypomethylation progresses significantly from primary to metastatic cancer and defines a poor prognostic signature in four different gynaecological cancers. Finally, we associate expression of TET enzymes, which are involved in active DNA demethylation, to MESC hypomethylation in cancer. These findings have major implications for cancer and embryonic stem cell biology and establish the importance of systemic DNA hypomethylation for predicting prognosis in a wide range of different cancers. DNA methylation is an important chemical modification of DNA that can affect and regulate the activity of genes in human tissue. Abnormal DNA methylation and its subsequent effects on gene activity are a hallmark of cancer, yet when precisely these DNA methylation changes occur and how they contribute to the development of cancer remains largely unexplored. In this work we measure the methylation state of DNA at over 14,000 genes in over 1,475 samples, including normal and benign cells, invasive cancers, and metastatic cancer tissue. Using cervical cancer as a model, we show that gain of abnormal methylation at genes typically un-methylated in stem cells can be detected up to 3 years in advance of the appearance of pre-cancerous cells, while those genes typically methylated in stem cells lose this methylation progressively throughout cancer development. Furthermore, we discover that this process of methylation loss during cancer progression is a marker of poor disease outcome common to all four major women-specific cancers: breast, ovarian, endometrial, and cervical cancers. Finally we demonstrate the relationship between loss of methylation and cancer-specific over-production of a specific protein known to play an active role in removing methylation from DNA. Taken together these findings highlight the complex nature of DNA methylation dynamics in cancer development as well as their potential exploitation for clinical gain. Aberrant DNA methylation is one of the most important cancer hallmarks Both hyper and hypomethylation are commonly observed in cancer Another feature of the epigenetic landscape characterising human embryonic stem cells (hESC) was described by Lister et al Motivated by these outstanding questions, we decided to (i) explore the dynamics of epigenetic changes at stem cell loci (PCGTs and MESCs) throughout all stages of human carcinogenesis and (ii) to investigate their potential role in predicting poor prognosis.To address our first aim, we used as a model the uterine cervix, since screening programs in place allow easy access to this organ, and cervical carcinogenesis is also one of the few scenarios in humans where DNA methylation changes in the actual cell of origin and occurring throughout disease progression can be analyzed. Specifically, we measured DNA methylation at over 27,000 CpGs in cervical cells and at three different stages: (a) three years before onset of dysplastic changes, (b) at the stage of non-invasive dysplasia, and (c) at the stage of invasive cervical cancer. To address our second aim we analysed DNA methylation data from 5 independent cohorts encompassing a total of 1,026 tumour samples in 4 different gynaecological cancers. In total, we analysed DNA methylation data from 10 independent studies, encompassing normal and cancer tissue from 5 different tissue types, including metastases .Using these data we here report four major novel aspects of cancer epigenetics: (i) Hypermethylation at PCGT stem cell loci occurs up to three years before the first signs of morphological transformation, (ii) hypomethylation at MESC stem cell loci is a hallmark of cancer invasion, affecting both epithelial and stromal compartments, and increases further in metastases, (iii) hypomethylation instability at MESCs defines a stem cell DNA methylation signature that predicts poor prognosis in multiple human cancers independently of standard prognostic factors, and (iv) expression of TET enzymes All methylation data in this study were generated with the Illumina Infinium Human Methylation27 beadchip array , which aafter three years (cases), whereas the other 77 remained normal (controls). These samples were matched for age and HPV status. (2) ‘Dysplasia (Dy)’: age-matched non-invasive dysplastic epithelial cells (CIN2/3)  and normal cervical epithelial cells  collected within screening programs To investigate the dynamics of DNA methylation in human carcinogenesis we designed a study with samples from three different phases reflecting cervical carcinogenesis: (1) ‘Before Dysplasia (BDy)’: normal cervical epithelial cells collected within the ARTISTIC trial −100), especially for those PCGT CpGs located within PMDs . We verified that PCGT enrichment was also independent of HPV status (P<0.005 for HPV+ and HPV−). Notably, the frequency of hypermethylation remained fairly constant throughout the phases from non-invasive dysplasia to invasive cancer , non-invasive dysplastic samples had an increased MESC enrichment in hypomethylated CpGs  and eventually MESC CpGs contributed most significantly to hypomethylated CpGs in invasive cancer  commonly hypomethylated at a frequency of at least 10% in all four cancers (P<10−4), there were substantially larger overlaps between related cancers such as ovarian and endometrial cancer , P = 3.2×10−124). Gene Set Enrichment Analysis (GSEA) The ability of the DeMI index to predict clinical outcome in multiple cancers indicates that a core set of MESC CpGs may be involved. To investigate this we ranked the MESC CpGs according to the frequency of hypomethylation in each of the cancers considered. Many CpGs were observed to be hypomethylated in large fractions of tumours . While tUp until recently it has been assumed that DNA demethylation in cancer is a passive event, occurring as a result of absent re-methylation during DNA replication, with a consequent dilution of this covalent DNA modification. This view has now been substantially challenged by the identification of TET (ten eleven translocation) dioxygenases, which can convert 5-methylcytosine into 5-hydroxymethylcytosine and 5-carboxylcytosine, which thus constitutes a pathway for active DNA demethylation Epithelial cells of the uterine cervix offer a unique opportunity to study epigenetic alterations throughout carcinogenesis. Our first key result is the demonstration that normal cells of origin acquire methylation changes at least three years in advance of the first morphological changes. Specifically, our data demonstrate that PCGT hypermethylation and MESC hypomethylation are major contributors to early cervical carcinogenesis. This is independent of human papillomavirus (HPV) infection as our study was matched for HPV status, and since PCGT enrichment was observed in both HPV+ and HPV− samples. Importantly, the observed enrichments were also independent of the levels of methylation in normal tissue. That is, MESCs which showed full methylation  or hemi-methylation  were preferentially hypomethylated in dysplasia and cancer in comparison to control sets of CpGs with same methylation levels in normal tissue.BRCA1 mutation carriers, suggesting that BRCA1 is an important regulator of the DNA methylome and that aberrant BRCA1 function could lead to increased predisposition to cancer through increased methylation at PCGT loci. The fact that BRCA1 mutation carriers showed increased PCGT methylation in their blood cells but are at no substantial increased risk to develop blood-borne cancers suggests that PCGT hypermethylation refers a substantial risk but that there are additional factors required .The role of PCGT methylation as a very early event is further supported by our finding that PCGTs were highly enriched among CpGs which were hypermethylated in blood cells from Our second key result is that MESC hypomethylation occurs in both the epithelial and stromal components of cancer and that this is a progressive process, increasing significantly towards invasion and metastatic cancer. This in turn suggests that the level of MESC hypomethylation in primary tumours may be an important determinant of clinical outcome.Indeed, our third key result is the report of a stem cell (MESC) DNA hypomethylation signature that can predict clinical outcome in multiple human cancers, independently of known prognostic factors. To the best of our knowledge this constitutes the first report of a common prognostic signature in cancer that is based on DNA methylation, and is therefore an epigenetic analogue to the prognostic genomic instability signature presented in Besides the key distinction of PCGT and MESC CpGs, we also observed that the localisation of CpGs in relation to PMDs was another important facet of the pattern of DNA methylation changes. Specifically, PCGT hypermethylation was observed preferentially within PMDs, while the progressive MESC hypomethylation in cancer was equally strong in PMDs and non-PMDs. We point out that while the PMDs considered here were defined for colon cancer cells, that these broad regions of partial methylation overlap significantly between colon tissue and fibroblasts, suggesting that these regions may be largely similar also between different tissues.The similarities between normal developmental and cancer epigenetic programming are intriguing. While embryonic stem cells suppress differentiation-inducing genes reversibly via promoter occupancy of PRC2, cancer cells suppress these same genes much more robustly via covalent DNA modification. Even more interestingly, trophoblast cells whose core function is to invade the maternal tissue and form the placenta, are relatively more hypomethylated compared with the inner cell mass, which will differentiate into the embryo It could also be argued that the other DNA methylation changes we have reported here are the result of changes in the stromal and immune cell compartments of the tumours. However, we verified using Principal Components Analysis (PCA) and GSEA analysis Finally, the crucial role of TET3 in DNA demethylation and early development, its overexpression in cancer, and the observed correlation with MESC hypomethylation, supports the view that aberrant developmental programs leading to reprogramming of the epigenome in adult cells may be critical for carcinogenesis. Interfering with these aberrant programs may therefore lead to novel ways to treat cancer.In summary, our findings suggest that epigenetic deregulation of two distinct sets of genes, both important for stem cell integrity, impact carcinogenesis in different ways: one process involves gain of methylation and is a hallmark of de-differentiation and early oncogenesis, while the other involves loss of methylation and is a key determinant of invasion and clinical outcome.A recent study used bisulfite sequencing to map, at single-base-resolution, DNA methylation throughout the majority of the human genome in both embryonic stem cells and fibroblasts PolyComb Group Target genes (PCGTs) were defined as CpGs which are occupied by SUZ12 and/or EeD and/or are trimethylated at Lysine 27 on histone H3 in human embryonic stem cells  and quantified via spectrophotometry  with 600 ng DNA from each sample. DNA from whole blood was extracted using a chloroform based extraction method from 400 µL of blood. All DNA samples were bisulphite modified using the EZ DNA Methylation Kit D5004/8  according to the manufacturer's instructions.The genome wide methylation analyses were performed using the validated Illumina Infinium Human Methylation27 BeadChip  Total RNA was isolated as previously described U and M values, β-values (as generated from the Beadstudio software) and built-in controls were used to evaluate the quality of individual arrays. Samples with low BS conversion efficiency  were excluded, as well as other outliers that we detected using boxplots of total intensity I = U+M values and histograms of β-values. Samples were filtered further according to CpG coverage, using the Beadstudio P-values of detection of signal above background.Quality control procedures and intra-array normalisation were run on all data except the ‘Colon CA’, ‘Lung CA’, and ‘Ovarian CA’ sets, for which the intra-array normalised data was downloaded directly from Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases. Background corrected Enrichment analysis was performed using a two-tailed Fisher's exact test. Odds ratios (OR) and 95% confidence intervals (CI) of enrichment were also computed and their corresponding significance levels estimated. Enrichment analysis was performed with a range of thresholds to check for robustness and using the Infinium 27 k array as reference to avoid array-specific bias.A linear regression approach was used to model the association between disease status (cases or controls) and the CpG β-value methylation profile. Adjustment for age and experimental factors (e.g. bisulphite conversion) was performed by inclusion of these factors in the model as covariates. Chip effects were observed, and in this study all data were adjusted by either applying the “ComBat” method  Given the two disease-status-associated CpG lists (hyper- or hypomethylated) obtained from the supervised analysis, the two-tailed binomial test was used to detect the skewness of the methylation in various categories  of the CpGs  and S2.We devised an Epigenetic Instability Index (EpI) for each tumour sample as follows. First, CpG readings were defined as unmethylated (0) , hemimethylated (1) , and methylated (2) . Next, we selected CpGs with stable methylation profiles in normal tissue, defined as those CpGs with the same methylation state in all normal samples corresponding to the given tissue. These stable CpGs can undergo four types of DNA methylation changes in cancer: 0->1/2, 1->2, 1->0 and 2->0/1. Therefore, for each tumour sample, we computed four different “instability” indices, reflecting the fraction of stable CpGs undergoing the specific types of DNA methylation changes shown. When computing these indices, and to ensure their robustness to the choice of methylation thresholds above, we also required at least a 10% change in β-values for calling DNA methylation differences between normal and cancer tissue. This buffering therefore avoids calling potentially small differences in β-values (<10%), which nevertheless may trespass the methylation thresholds  used. The EpI indices were also computed by restricting the set of stable CpGs to those mapping to PCGT and MESC stem cell loci. Since the great majority of PCGT CpGs were observed to be stably unmethylated (0) in normal tissue, this resulted in 3 “stem cell EpI” indices: PCGT (0->1/2), MESC (1->0), MESC (2->0/1). We call the latter index the Demethylation instability index (DeMI).Univariate and multivariate Cox regression models were used for the survival analysis. In the multivariate analysis, besides DNA methylation β-values (or the EpI index), those clinical and histological factors, which were associated with survival in univariate analysis were also included as covariates.Figure S1Differential dynamics of hypermethylated and hypomethylated PMD PCGTs and PMD MESCs. Bar charts representing percentages of significantly hypermethylated (blue) and hypomethylated (orange) PMD PCGT and PMD MESC CpGs in (A) each stage of cervical carcinogenesis: Cervix ‘Before Dysplasia’, ‘Dysplasia’, and ‘Invasive Cancer’, all relative to normal cervix tissue; and in (B) ‘Breast CA’, ‘Endo CA’, ‘Colon CA’, and ‘Lung CA’, all relative to their respective normal controls. The significance of the binomial test assessing skew of hypermethylated versus hypomethylated ( and S4) is indicated by ‘*’, ‘**’, and ‘***’ for P-value<0.05, 0.01, and 0.001 respectively.(TIF)Click here for additional data file.Figure S2Differential dynamics of hypermethylated and hypomethylated nonPMD PCGTs and nonPMD MESCs. Bar charts of the percentages between the disease (or mutation) status associated hypermethylated (blue) and hypomethylated (orange) for nonPMD polycomb group target gene (PCGT) CpGs and nonPMD methylated in human embryonic stem cells (MESC) CpGs that pass their corresponding significance level thresholds (the same notation as in (TIF)Click here for additional data file.Figure S310 that test the association with the cases and controls of the cervix ‘Before Dysplasia’ (CIN2/3 status), ‘Dysplasia’ (CIN2/3 status), and ‘Invasive Cancer’ (cancer status) on the PMD PCGT (left column) and PMD MESC (right column) CpGs. Green vertical lines denote the significant level thresholds of P-value = 0.1 for ‘Before Dysplasia’ and ‘Dysplasia’, and 0.001 for ‘Invasive Cancer’. The overall numbers of CpGs that are hypermethylated (blue) and hypomethylated (orange) with their associated two-sided Binomial test P-value are given on the left hand side of the P-value threshold lines and the number of CpGs that are hypermethylated (blue) and hypomethylated (orange) pass the corresponding P-value threshold with their Binomial test P-value on the right.Statistical output from linear regression model estimating the association of the PMD PCGT and PMD MESC CpGs to the outcomes of the three stages of cervical carcinogenesis. Scatterplots of the linear regression fitted  t-statistics against their corresponding −log(TIF)Click here for additional data file.Figure S4Statistical output from linear regression model estimating the association of the nonPMD PCGT and nonPMD MESC CpGs to the outcomes of the three stages of cervical carcinogenesis. Scatterplots of three cervical sets, similar to (TIF)Click here for additional data file.Figure S5Enrichment analysis of PMD PCGTs and PMD MESCs in the hyper- and hypomethylated cervical cancer CpGs. Cumulative enrichment analysis  of PCGTs among CpGs unmethylated  in normal cervix and which become hypermethylated in (i) normal samples three years prior to dysplasia (BDy), (ii) non-invasive dysplastic samples (Dy), and (iii) invasive cervical cancer (CA) in PMDs (A) and nonPMDs (B) respectively. Similarly, enrichment of MESCs among CpGs methylated  in normal cervix and that become hypomethylated in cases in PMDs (C) and nonPMDs (D) respectively.(TIF)Click here for additional data file.Figure S610 testing the association with the cases and controls of ‘Breast CA’ (cancer status), ‘Endo CA’ (cancer status), ‘Colon CA’ (cancer status), ‘Lung CA’ (cancer status), and ‘BRCA1 MUT’ (BRCA1 status) on the PMD PCGT and PMD MESC CpGs. Green vertical lines denote the significant level thresholds of P-value = 0.1 for ‘BRCA1 MUT’ and 0.001 for all the others. The overall number of CpGs that are hypermethylated (blue) and hypomethylated (orange) with their associated two-sided Binomial test P-value are given on the left hand side of the P-value threshold lines. The number of CpGs that are hypermethylated (blue) and hypomethylated (orange) pass the corresponding P-value threshold with their Binomial test P-values on the right.Statistical output from linear regression models estimating the association of the PMD PCGTs and PMD MESC CpGs to outcomes in five cohorts. Scatterplots of the linear regression fitted  t-statistics against their corresponding −log(TIF)Click here for additional data file.Figure S7Statistical output from linear regression models estimating the association of the nonPMD PCGT and nonPMD MESC CpGs to the outcomes in five cohorts. Scatterplots of five cohorts, similar to (TIF)Click here for additional data file.Figure S8Magnitude of the anti-correlation between hypomethylated cervical cancer CpGs and TET mRNA. Hypomethylated MESCs are significantly higher anti-correlated with TET2 and TET3 mRNA expression levels than the hypomethylated nonMESCs in the cervical cancer samples. P-values are obtained from the Wilcoxon one-sided test.(TIF)Click here for additional data file.Figure S9Magnitude of the anti-correlation between hypomethylated cervical cancer CpGs (grouped) and TET mRNA. Higher anti-correlated signature with TET2 and TET3 mRNA expression levels among hypomethylated MESCs than hypomethylated nonMESCs in the cervical cancer samples independent from the chosen baselines of the methylated and hemimethylated CpGs . P-values are obtained from the Wilcoxon one-sided test.(TIF)Click here for additional data file.Figure S10TET mRNA expression level comparison between the normal cervix and cervical cancers. Boxplots of TET1, TET2.1, TET2.2 and TET3 mRNA expression levels of the normal cervix and cervical cancers. P-values are obtained from the Wilcoxon two-sided test.(TIF)Click here for additional data file.Table S1The MESC CpGs mapped to 27 k Infinium array. List of the 5,943 MESC CpGs that mapped to the Illumina Infinium Human Methylation27 beadchip array with the information of their IlluminaID, geneID, gene symbol, MapInfo, and chromosome.(XLS)Click here for additional data file.Table S2The PCGT CpGs mapped to 27 k Infinium array. Similar to (XLS)Click here for additional data file.Table S3Summary tables of the uni- and multivariate Cox regression model analysis of the PCGTs and MESCs Epigenetic Instability Index (EpI) in endometrial, breast, ovarian, and cervical cancers. Univariate (UV) and multivariate (MV) Cox regression results for the PCGTs and MESCs EpI in endometrial, breast, ovarian and cervical cancer overall survival (OS) and relapse free survival (REL) with number of samples (n), Hazard ratio (HR), 95% confident interval, and P-value (P).(XLS)Click here for additional data file.Table S4PCGT and MESC enrichment analysis amongst hypermethylated and hypomethylated cancer prognostic CpGs. Enrichment analysis (Fisher's exact tests odds ratios (OR), 95% confidence intervals (CI) and P-values (P)) of PCGTs (colon-PMD and nonPMD) and MESCs (colon-PMD and nonPMD) among the top 500 hypermethylated and hypomethylated cancer  prognostic CpGs.(XLS)Click here for additional data file.Table S5Overlap between MESC CpGs with stable methylation levels in normal tissue and that become hypomethylated in cancer with the top ranked 1,000 prognostic MESC CpGs that are hypomethylated in poor outcome samples in endometrial, breast, ovarian, and cervical cancers.(XLS)Click here for additional data file.Table S6MESC CpGs according to the frequency of hypomethylation  as defined by the demethylation (2->0/1) instability index (DeMI) in the endometrial, breast, ovarian, and cervical cancers.(XLS)Click here for additional data file.Table S7GSEA results of MESC CpGs with a frequency of hypomethylation (defined by DeMI index) in cancer of at least 5%  and of at least 10% in cervical cancer.(XLS)Click here for additional data file.Text S1A detailed description of the definitions, study population, materials, and primers used in this study is provided.(DOC)Click here for additional data file.


All three selections were successful, with large differences in haloperidol response emerging within three generations. Using a custom differential network analysis procedure, we found that gene coexpression patterns changed significantly; importantly, a number of these changes were concordant across genetic backgrounds. In contrast, absolute gene-expression changes were modest and not concordant across genetic backgrounds, in spite of the large and similar phenotypic differences. By inferring strain contributions from the parental lines, we are able to identify significant differences in allelic content between the selected lines concurrent with large changes in transcript connectivity. Importantly, this observation implies that genetic polymorphisms can affect transcript and module connectivity without large changes in absolute expression levels. We conclude that, in this case, selective breeding acts at the subnetwork level, with the same modules but not the same transcripts affected across the three selections.We performed short-term bi-directional selective breeding for haloperidol-induced catalepsy, starting from three mouse populations of increasingly complex genetic structure: an F Haloperidol is a member of a class of drugs denoted as “typical” or first-generation antipshychotics since their adoption as psychosis treatments. A number of locomotory side effects, including dyskinesia, complicate their use in humans. Mice injected with haloperidol develop catalepsy  and this response is considered a model for human locomotory disturbances. Mouse inbred strains differ widely in their response to haloperidol, signaling a strong genetic component for this trait; a number of associated genetic loci have been previously detected in mouse crosses 50. The lines also differed in their response to structurally dissimilar D2 dopamine receptor antagonist, raclopride, strongly suggesting that the basis for selection was pharmacodynamic and not pharmacokinetic. Selection produced only modest changes in striatal gene expression despite the use of large populations (N =  >40/line). In contrast, applying Weighted Gene Coexpression Network Analysis (WGCNA) 2 intercross formed from the B6 and D2 strains, and the other was the HS-CC that was formed from the eight founder strains of the Collaborative Cross (CC). Using single nucleotide polymorphisms (SNPs) as a surrogate for genetic diversity, the F2 intercross is 50% less diverse than the HS4, and the HS-CC is three times more diverse We have examined the striatal gene-expression covariance structure in animals that were selectively bred from a heterogeneous stock (HS) for haloperidol response and non-response All animal care, breeding and testing procedures were approved by the Laboratory Animal Users Committees at the Veterans Affairs Medical Center, Portland, OR, and at the Oregon Health & Science University, Portland, OR.The formation of the HS colonies was described previously http://www.r-project.org), and outlier samples were removed. Probes not expressed above background (p<0.01) in at least a quarter of the samples were removed. For each probe, we computed the coefficient of variability (CV) and we selected for network construction the top 50% most variable probes. The intersection of these probes across the three datasets resulted in 6755 probes. The data were quantile normalized. Microarray datasets are publicly available in the Gene Expression Omnibus database Our data pre-processing steps closely follow procedures described previously 2 and HS4 selected lines were genotyped using a panel of 768 SNPs previously described http://www.neogen.com/GeneSeek/SNP_Illumina.html). Genome-wide genetic differences were analyzed following the AMOVA procedure Fij between transcripts xi and xj then becomes aij = |corr|β; β = 10 was selected in accordance to the scale-free topology criterion We constructed the coexpression network using the WGCNA approach aobs corresponds to preservation for a given network statistic and module, μa and σa correspond to mean and standard deviation of preservation values, in our case generated from the empirical distribution of preservation values between mixed-sample networks. While for module preservation the Z scores are positive, signifying that modules are more preserved than random groups of nodes, disruption Z scores are often negative. The negative Z values result from the fact that preservation between two networks corresponding to different biological conditions is lower than preservation between two networks created using a mixture of samples: often aobs is smaller than μa. Following Recent work The HS-CC dense genotype data, which included genotypes of the eight parental strains, was used to infer the probability of each ancestral allele for each combination of genomic interval and each individual. We used a dynamic programming algorithm available as part the R package HAPPY 2, HS4 and HS-CC) were phenotyped for haloperidol response using a two-step process that resulted in assigning animals to one of four groups; “1” was the least responsive, and “4” was the most responsive. Breeding pairs were selected from the most extreme response groups. The selection and breeding were continued for two additional generations; in the third generation, parents were bred for three rounds to produce progeny for gene expression. As illustrated in Procedures for testing and selecting mice have been described previously 2 and HS4 founders and their respective selected lines were genotyped using a SNP panel described elsewhere -5).The Fwww.bioconductor.org) and further adjusted using the False Discovery Rate (FDR) 2 and HS4 selections . There were no differentially expressed transcripts common to all three selections. The gene transcripts showing a significant differential effect are found in Dataset S1, in Differential striatal gene expression for the three pairs of selected lines was calculated using the eBayes modified t-statistic available in Bioconductor  was constructed using a mixture of High and Low samples; differences in connectivity values were used to construct an empirical distribution of connectivity changes that can occur by chance. Differences between the High and Low networks were normalized against the empirical distribution. Using this procedure, each transcript connectivity change between High and Low networks was assigned a Z score; scores Z>2 were considered significant. We found a relatively large number of transcripts significantly changed network connectivity: 458 (7.0%), 499 (7.6%) and 1537 (23.4%) in F2, 12 in HS4 and 21 in HS-CC; a complete listing of changes in network statistics is available in Dataset S5, in We also evaluated the collective, cumulative change in connectivity of all transcripts within a module. As detailed in Methods, we used an adapted version of the module preservation procedure that is part of the WGCNA pipeline Myo5a, falls within the range of a previously detected QTL for haloperidol induced catalepsy Rps6ka5 has been previously associated with dopamine processing within striatal neurons Spock1 has been previously associated with genetic effects on tyrosine hydroxylase levels within dopaminergic neurons Bcl11b, has been previously shown to strongly influence striatal gene expression Bcl11b to illustrate connectivity and allelic differences in The dense genotyping of the HS-CC samples facilitated the integration of genetic and coexpression data. We identified genomic regions with significant allele distribution differences between the High and Low selected lines. By combining the genotype data from the founder inbred lines, it was possible to infer, for each animal, the most probable ancestral alleles for each genomic interval 2-like dopamine receptors), the striatum is the major target for the extra-pyramidal response and the phenotype is highly heritable 2 receptor density, the relative amounts of the long and short receptor isoforms or haloperidol pharmacokinetics 2–D3 receptor antagonist, raclopride, shows the same inbred strain distribution pattern 1 receptor antagonists, e.g., SKF 23390 Haloperidol-induced catalepsy is an ideal phenotype for integrative analysis of gene networks, genetic diversity and behavior. The mechanism of drug action is well established  specifically and networks generally may be the key to understanding heritability, how best to investigate the gene-behavior relationship, especially with a translational perspective? Here, we simply offer one approach. Some behaviors, especially those that substantially involve subcortical circuitry, have a large number of relevant isomorphic animal models. With these models in hand, it is possible to use network techniques to detect key modules; the translational goal is to determine which genes within the key module(s) are targets for manipulation and behavioral modification. We argue that the inclusion of the HS-CC or a related population such as the Diversity Outcross Drd2 (“Pink”), these include Rgs9, Pde10a and Chat , Tcf4 (“Green”) and Lrrk2 and Foxp2 (“Grey 60”) Jmjd1a and Jmjd2b) are key hub genes , perhaps suggesting an epigenetic strategy for modifying haloperidol response.Another target driven approach is to focus on the “hub” genes. From the scale-free network perspective, hub disruption will have the greatest affect on modular connectivity. It is not necessary that the hub targets be genes that are affected by selection although focusing on these genes provides insight into the causes of natural variation. In the three selection consensus modules, there are a number of hub genes previously shown to significantly affect locomotor behavior and in some cases haloperidol response. In addition to Overall, the data presented here argue that a systems biology approach is needed, at least in some contexts, to investigate the relationships between genes and behavior. The results from the three independent selections illustrated that at the gene transcript level, neither differential expression, nor differential connectivity in one selection population predicted similar results in another selection population; however at the module level, connectivity changes did overlap. Extracting module-dependent information requires relatively large gene expression datasets, which in turn facilitate an accurate assessment of the covariance structure. This assessment can be improved by substituting RNA-Seq for microarray based strategies File S1Contains: Dataset1: Gene transcripts showing a significant differential effect. Dataset2: Gene Ontology (GO) annotation of the consensus modules. Dataset3: Transcription factor binding sites (TFBS) enrichment of consensus modules. Dataset4: Transcription factor binding site pair enrichment of consensus modules. Dataset5: Network changes quantified using Z scores. Dataset6: Gene transcripts with significant changes in both expression and connectivity.(ZIP)Click here for additional data file.File S2Supplementary Methods: Definition of network statistics used in the analysis.(DOCX)Click here for additional data file.


Dried amniotic membrane (AM) can be a useful therapeutic adjunct in ophthalmic surgery and possesses logistical advantages over cryopreserved AM. Differences in preservation techniques can significantly influence the biochemical composition and physical properties of AM, potentially affecting clinical efficacy. This study was established to investigate the biochemical and structural effects of drying AM in the absence and presence of saccharide lyoprotectants and its biocompatibility compared to cryopreserved material.AM was cryopreserved or dried with and without pre-treatment with trehalose or raffinose and the antioxidant epigallocatechin (EGCG). Structural and visual comparisons were assessed using electron microscopy. Localisation, expression and release of AM biological factors were determined using immunoassays and immunofluorescence. The biocompatibility of the AM preparations co-cultured with corneal epithelial cell (CEC) or keratocyte monolayers were assessed using cell proliferation, cytotoxicity, apoptosis and migration assays.in vitro. Cellular health assays showed that dried AM with trehalose or raffinose are compatible and superior substrates compared to cryopreserved AM for primary CEC expansion, with increased proliferation and reduced LDH and caspase-3 levels. This concept was supported by improved wound healing in an immortalised human CEC line (hiCEC) co-cultured with dried and trehalose or raffinose membranes, compared to cryopreserved and fresh AM.Drying devitalised AM epithelium, but less than cryopreservation and cellular damage was reduced in dried AM pre-treated with trehalose or raffinose. Dried AM alone, and with trehalose or raffinose showed greater factor retention efficiencies and bioavailability compared to cryopreserved AM and demonstrated a more sustained biochemical factor time release Our modified preservation process and our resultant optimised dried AM has enhanced structural properties and biochemical stability and is a superior substrate to conventional cryopreserved AM. In addition this product is stable and easily transportable allowing it to be globally wide reaching for use in clinical and military sectors. Amniotic membrane (AM) is the inner most extraembryonic membrane that surrounds the foetus in a sac of amniotic fluid, functioning as a protective barrier to ascending infection and trauma during pregnancyAM has proven to be a highly versatile surgical adjunct. The clinical use of AM was first reported in the treatment of skin wounds, in 1910Therapeutic AM is extensively used in ophthalmic surgery and was first applied with chorion, as a replacement for scarred conjunctival tissueAM has been shown to act as a scaffold for cell growthet alIn many countries, AM is obtained from elective caesarean section deliveries and typically frozen in medium containing glycerol or dimethylsulphoxide (DMSO) while the donor is screened for a spectrum of infectious diseases. The effects of DMSO and glycerol preservatives on the structural and biochemical integrity of cryopreserved AM are unclearConventional freeze-drying requires the tissue to be frozen prior to drying. In AM, this appears to result in structural freeze damage and subsequent factor loss as compared with conventional cryopreservation techniquesSterile, dried AM can be prepared in the presence of complex saccharide lyoprotectants such as trehaloseIn this study, we investigated the structural and biological properties of dried AM, and its biocompatibility as an ocular surface dressing, using cryopreserved AM as a comparator. In addition we pre-treated AM with trehalose or raffinose and EGCG to further augment the tissue quality.The following research was carried out with the approval of the Nottingham Research Ethics Committee and the study (OY110101) complied with the tenets of the Declaration of Helsinki. Written informed consent from the donor (participant age range 25–37) or the next of kin was obtained for sample use in this research project and prepared according to previously published methodologyTo optimise the preservation process, prior to drying, AM segments were uniformly spread out in Petri dishes and bathed in a series of solutions containing glycerol/PBS (ratios of 1∶4 and 1∶2), DMSO (Sigma-Aldrich)/phosphate buffered saline , and tertiary butyl alcohol /PBS (10 and 40% v/v) for 10 minutes each. Alternatively segments were incubated with 2, 10, and 25% w/v D- (+)-Trehalose dihydrate  in PBS or 25, 100 and 200 mM w/v D-(+)-Raffinose pentahydrate (Acros Organics), and EGCG  for 2 hours at 37°C.Processed AM segments from 12 donors were either used fresh or prepared and preserved using one of five defined methodologies.AM segments were preserved in sterile Dulbecco's PBS (Sigma-Aldrich) in 20 mL sterile tubes, at −80°C, using established methodologies within our departmentFresh segments were uniformly spread out, epithelial side up, in vacuum pouches. This was then double heat sealed on three sides prior to drying. Drying was performed using an Alpha 1–4 LSC freeze-dryer . Prior to sample drying the ice condenser was equilibrated to −45°C. This allows any vapour present in the drying chamber to be removed during the drying procedure, by freezing to the condenser itself. The drying cycle comprised of a main dry phase for 1 hour  followed by a final drying phase for 30 minutes . Following completion of the drying cycle the pouch was heat sealed under vacuum using a Multiple 315 vacuum packaging chamber,  and stored at room temperature, and away from direct light, until further analysis.AM segments were denuded of epithelium with thermolysin  according to our published methodologiesAM was pre-treated with 10% w/v D- (+)-Trehalose dihydrate and EGCG -Raffinose pentahydrate and EGCG (1 mg/mL) for 2 hours at 37°C. Prior to drying membrane sections were washed briefly in a 1∶10 dilution of the original raffinose solution to remove excess residue.Transparency of the pre-treated and dried membranes were assessed by taking images against printed material and a dark granulated background and compared to non-treated dried only control material. Membranes were graded according to a 3 point scale with control material being assigned the maximum of 3 for optimal transparency; this was carried out by two separate investigators.Scanning EM (SEM) and transmission EM (TEM) studies were performed on membranes from 3 separate donors, prepared in duplicate following different preservation techniques. For each sample, 1 cm diameter discs of preserved AM samples were overlaid on corresponding discs of polyvinylidene fluoride (PVDF) membrane  and processed for SEM and TEM according to previously published methodologiesTX) buffer for 20 minutes at room temperature. Insoluble material was removed by centrifuging at 20,000×g, for 15 minutes, at 4°C and the protein concentration of each supernatant was determined using a 2D Quant kit . Protein arrays were carried out in duplicate using SearchLight immunoassay technology  for a profile of 48 protein analytes. Analytes failing to provide a data value were omitted from the study and remaining data normalised according to ng/mg of protein extract.Soluble proteins were extracted from segments of fresh, cryopreserved, denuded, dried, trehalose or raffinose treated membranes from 3 separate donors. Samples were ground under liquid nitrogen and reconstituted in 1× Tris buffered saline +0.05% v/v Triton-X (Sigma-Aldrich) (TBSDifferently preserved AM sections (5 cm Ø) from 3 separate donors were washed in 5 mL saline containing protease inhibitors  for 3×10 minutes. Cryopreserved AM storage medium and washes were concentrated and retained for analysis. Samples were concentrated using Amicon® centrifugal filter devices  and protein concentrations determined as previously described. EGF and TGF-β1 concentrations were determined using ELISA duo kits  with microplates pre-coated with monoclonal antibodies specific for the human markers in question, as per manufacturer's instructions. Sample absorbances were read at 450 nm and concentrations were then calculated from a standard curve of known values, and subsequently normalised against protein concentration.Triplicate AM sections from 3 donor samples were preserved as previously described and stored in sterile vacuum pouches at ambient temperature and away from direct light, for a period of 4, 8, 12, 24, 48 and 60 weeks. Following storage, soluble proteins were extracted from the tissue sections and protein concentrations determined using a 2D Quant kit and EGF and TGF-β1 concentrations assessed using ELISA kits previously described.AM sections from 3 donors for each sample preservation type were prepared and immunostained according to published methodologiesCells were isolated according to a previously published methodology2 and filtered with a 41-µm nylon filter , to remove non cellular debris. Culture medium was added to the collagenase filtrate solution prior to centrifugation at 450×g for 6 minutes. The cell pellet was resuspended in M199 basal culture medium (Sigma-Aldrich) supplemented with 20% v/v heat-inactivated FBS (Fisher Scientific), 2.5 µg/ml Plasmocin™, 0.02 µg/ml gentamicin, 0.5 ng/ml amphotericin B, and 1.59 mM L-glutamine (Sigma-Aldrich).Keratocytes were isolated according to a previously published methodologyhiCEC . 15 mm membrane discs were laid over the support and held in place with an outer ring. The support was then inverted in a 24-well plate so it was immersed in the media but not in direct contact with the cells. Direct cultures were assembled by seeding the cells directly on top of the AM/CellCrown™ supports. Cells were seeded at 0.05×10Factor time release studies were carried out using the above system apart from the membranes were submersed in sterile PBS. Samples of PBS (120 µl) were taken following 1, 2, 4 and 10 days in culture. Samples were stored at −80°C prior to SearchLight protein array analysis and EGF and TGF-β1 ELISA experiments.in vivo Cell-8 assay (Sigma-Aldrich), cytoxicity was measured using a lactate dehydrogenase (LDH) enzyme based assay  and apoptosis was determined using a caspase-3 colorimetric assay . All assays were performed according to manufacturer's protocols. Levels were calculated by subtracting cell only control values for each day and results are expressed as a percentage increase or decrease relative to the previous day.Cell proliferation was assessed using the A scratch wound closure assay was performed five days post seeding, on confluent cultures starved of serum and EGF for 24 hours. A standard single linear scratch with a defined length of 1.6 cm was created in the cell monolayers across each well using a 10 µl pipette tip, giving a 300 µm wound width. Unattached cells were washed away and medium was replaced with media containing EGF and FBS. Wounds were photographed immediately (day 0) and then 2, 4, 6 and 10 days, at four pre-determined positions by phase-contrast imaging at 100× magnification. Wound healing for each culture was reported as the average linear speed of the wound edge closure over a 10 day period, using ImageJ software .U test and p<0.05 was considered significant.Results are presented as mean ± SEM. Statistical analyses were performed using the nonparametric Mann-Whitney Visual assessment of the differently preserved AM substrates, summarised in Following cryopreservation the typical amniotic epithelial cell (AEC) polygonal shape and cell patterns were lost. AEC appeared mainly intact but heavily damaged with clear fissures present between the cells with the damage and absence of surface microvilli . AEC hadin situ localisation and solubility state. This generated 5 specific sub-divisions of cohorts; (i) epithelial factors that were predominantly soluble, reduced with epithelial damage and therefore susceptible to preservation related stripping and removed with denuding; (ii) epithelial, predominantly insoluble factors not reduced by preservation but removed with denuding; (iii) stromal factors that were partially soluble, reduced by preservation but not by denuding; (iv) stromal and predominantly insoluble factors not reduced by preservation and proportionately increased following denuding and (v) factors that were undefined and considered to be both epithelial and stromal. Of the 45 protein factors analysed  proliferation was tested in a similar manor. Indirect culture showed a progressive increase in proliferation in the presence of dried, trehalose and raffinose treated AM compared to cryopreserved AM, with trehalose and raffinose AM promoting greater rates of growth. Increasingly negative proliferation rates were observed with denuded AM .Indirect culture of cells with dried, trehalose and raffinose treated AM demonstrated generally reduced cell death in both hiCEC and pCEC  validatiIndirect culture of dried preparations with CEC or pKer promoted no significant increases in caspase-3 activity compared to cell only controls . MarginaDirect culture promoted a greater and comparable increase in proliferation in both hiCEC and pCEC using dried, trehalose and raffinose treated AM , compareConversely, direct culture of pKer with all membrane preparations resulted in considerable negative effects on proliferation, supporting the evidence that AM inhibits pKer growthDirect culture of all samples types showed a moderate increase in cytoxicity after the first (hiCEC) and second (pCEC) day, with the greatest cytoxicity observed with cryopreserved AM and least so in dried, trehalose and raffinose treated AM . The incCytoxicity in direct cultures of both CEC cell types appeared to follow a pattern, which is most clearly seen in hiCEC, in that following an initial reduced cytoxicity on the first day (first two days and at a greater reduction for pCEC) a sharp increase in cytoxicity appeared to consistently occur on day 3 which then decreased over the remaining culture period . This paCryopreserved AM significantly (p<0.05) increased caspase-3 activity when directly cultured with hiCEC (0.4 fold), CEC (0.6 fold) and pKer (0.4 fold), compared to cells alone, following five days in culture . This suTo evaluate the effects of the AM substrates on re-epithelialisation following injury, scratch wound healing assays were performed and wound closure rates calculated at days 2, 6 and 8 compared to control (day 0) and in the presence of hiCEC indirectly cultured with the different AM preparations . ResultsIn recent years freeze-dried AM has become an alternative to cryopreserved AM as a substrate for stem cell expansionTo minimise the effects of freeze damage, we pre-treated AM with lyoprotectants prior to drying. Our SEM images show that while cryopreservation and drying without lyoprotectants are highly destructive to cellular integrity, our trehalose or raffinose treated AM preparations exhibited less visible structural damage and appear structurally akin to fresh AM. This was supported by TEM images showing that dried AM without pre-treatment had a more condensed collagen network, and the stromal and epithelial layers appeared thinner following the drying process. While the visual quality of reconstituted lyophilised AM has been reported to be poor in comparison to fresh AM and cryopreserved AMIn addition to the structural differences observed, our study confirmed that all preservation methods resulted in the loss of biological factors from AM.In 2009, Rodriguez-Ares and colleagues published similar results to our own findings. They analysed the biochemical profiles of AM preparations, concluding that the levels of biochemical factors associated with wound healing were decreased in lyophilised AM compared to cryopreserved, but these were not significantDifferences in preservation methods do not solely explain the variation in AM factor levels, which may also be influenced by gestational age, donor age, differences in handling techniques and the location of the sample's origin within the donor tissueAssuming that the retention of key growth factors in AM preparations is germane to its effectiveness in some clinical situations, the rate of release of these factors may also be significant. Ideally, AM would secrete its factors over a prolonged period and not its whole reserve instantaneously. Our assays show that as well as improved factor retention, the release of EGF from trehalose or raffinose treated AM was more sustained than cryopreserved AM over a 10 day period suggesting that the immediate release from cryopreserved was attributable to the freeze-thaw damage. This indicates that optimised trehalose and raffinose treated AM will have both increased factor content and prolonged release potentially improving the overall clinical effectiveness of AM over cryopreserved AM.per se.Cellular health assays supported this showing trehalose and raffinose treated AM substrates promote significant increases in corneal epithelial and keratocyte proliferation, with reduced overall cytotoxicity and apoptotic levels, compared to denuded and cryopreserved AM in both direct and indirect cultures. Similarities between cryopreserved and denuded AM further suggest that this phenomenon is explained by the removal of essential cell-based growth factors essential for promoting wound healing, rather than as a result of drying Increased wound closure rates were observed in hiCEC co-cultures with the optimised trehalose and raffinose treated AM substrates compared to cryopreserved and fresh. This may be attributed to the bioavailability of wound healing factors e.g. EGF, HGF, PDGF and TGF-β retained in the tissue by our optimised preservation technique. Growth factors are essential for the regulation of cellular processes including growth, proliferation and differentiation. If certain factor levels are diminished or absent this would have a detrimental effect on these processes and subsequent wound healing.In this study, we have shown that the saccharide lyoprotectants trehalose and raffinose improve the quality of dried AM by maintaining its structural and biochemical properties over extended periods. This improved stability will allow our optimised dried AM substrate to be stored and transported without the need for freezing, reducing costs and allowing it to be used as an ocular surface dressing outside modern hospital settings. In the absence of availability of optimised drying procedures our work indirectly supports the use of denuded AM, using an optimised procedure