Multipotent adult progenitor cells prevent functional impairment and improve development in inflammation driven detriment of preterm ovine lungs.

Background: Perinatal inflammation increases the risk for bronchopulmonary dysplasia in preterm neonates, but the underlying pathophysiological mechanisms remain largely unknown. Given their anti-inflammatory and regenerative capacity, multipotent adult progenitor cells (MAPC) are a promising cell-based therapy to prevent and/or treat the negative pulmonary consequences of perinatal inflammation in the preterm neonate. Therefore, the pathophysiology underlying adverse preterm lung outcomes following perinatal inflammation and pulmonary benefits of MAPC treatment at the interface of prenatal inflammatory and postnatal ventilation exposures were elucidated. Methods: Instrumented ovine fetuses were exposed to intra-amniotic lipopolysaccharide (LPS 5 mg) at 125 days gestation to induce adverse systemic and peripheral organ outcomes. MAPC (10 × 106 cells) or saline were administered intravenously two days post LPS exposure. Fetuses were delivered preterm five days post MAPC treatment and either killed humanely immediately or mechanically ventilated for 72 h. Results: Antenatal LPS exposure resulted in inflammation and decreased alveolar maturation in the preterm lung. Additionally, LPS-exposed ventilated lambs showed continued pulmonary inflammation and cell junction loss accompanied by pulmonary edema, ultimately resulting in higher oxygen demand. MAPC therapy modulated lung inflammation, prevented loss of epithelial and endothelial barriers and improved lung maturation in utero. These MAPC-driven improvements remained evident postnatally, and prevented concomitant pulmonary edema and functional loss. Conclusion: In conclusion, prenatal inflammation sensitizes the underdeveloped preterm lung to subsequent postnatal inflammation, resulting in injury, disturbed development and functional impairment. MAPC therapy partially prevents these changes and is therefore a promising approach for preterm infants to prevent adverse pulmonary outcomes.

Antenatal Ureaplasma Infection Causes Colonic Mucus Barrier Defects: Implications for Intestinal Pathologies.

Chorioamnionitis is a risk factor for necrotizing enterocolitis (NEC). Ureaplasma parvum (UP) is clinically the most isolated microorganism in chorioamnionitis, but its pathogenicity remains debated. Chorioamnionitis is associated with ileal barrier changes, but colonic barrier alterations, including those of the mucus barrier, remain under-investigated, despite their importance in NEC pathophysiology. Therefore, in this study, the hypothesis that antenatal UP exposure disturbs colonic mucus barrier integrity, thereby potentially contributing to NEC pathogenesis, was investigated. In an established ovine chorioamnionitis model, lambs were intra-amniotically exposed to UP or saline for 7 d from 122 to 129 d gestational age. Thereafter, colonic mucus layer thickness and functional integrity, underlying mechanisms, including endoplasmic reticulum (ER) stress and redox status, and cellular morphology by transmission electron microscopy were studied. The clinical significance of the experimental findings was verified by examining colon samples from NEC patients and controls. UP-exposed lambs have a thicker but dysfunctional colonic mucus layer in which bacteria-sized beads reach the intestinal epithelium, indicating undesired bacterial contact with the epithelium. This is paralleled by disturbed goblet cell MUC2 folding, pro-apoptotic ER stress and signs of mitochondrial dysfunction in the colonic epithelium. Importantly, the colonic epithelium from human NEC patients showed comparable mitochondrial aberrations, indicating that NEC-associated intestinal barrier injury already occurs during chorioamnionitis. This study underlines the pathogenic potential of UP during pregnancy; it demonstrates that antenatal UP infection leads to severe colonic mucus barrier deficits, providing a mechanistic link between antenatal infections and postnatal NEC development.

Identification of Compounds Protecting Pancreatic Islets against Oxidative Stress using a 3D Pseudoislet Screening Platform.

Oxidative stress leads to a lower success rate of clinical islet transplantation. Here, FDA-approved compounds are screened for their potential to decrease oxidative stress and to protect or enhance pancreatic islet viability and function. Studies are performed on in vitro "pseudoislet" spheroids, which are pre-incubated with 1280 different compounds and subjected to oxidative stress. Cell viability and oxidative stress levels are determined using a high-throughput fluorescence microscopy pipeline. Initial screening on cell viability results in 59 candidates. The top ten candidates are subsequently screened for their potential to decrease induced oxidative stress, and eight compounds efficient reduction of induced oxidative stress in both alpha and beta cells by 25-50%. After further characterization, the compound sulfisoxazole is found to be the most capable of reducing oxidative stress, also at short pre-incubation times, which is validated in primary human islets, where low oxidative stress levels and islet function are maintained. This study shows an effective screening strategy with 3D cell aggregates based on cell viability and oxidative stress, which leads to the discovery of several compounds with antioxidant capacity. The top candidate, sulfisoxazole is effective after a 30 min pre-incubation, maintains baseline islet function, and may help alleviate oxidative stress in pancreatic islets.

A Combination of Acetate, Propionate, and Butyrate Increases Glucose Uptake in C2C12 Myotubes.

BACKGROUND: Dietary fibers are subjected to saccharolytic fermentation by the gut microbiota, leading to the production of short chain fatty acids (SCFAs). SCFAs act as signaling molecules to different cells in the human body including skeletal muscle cells. The ability of SCFAs to induce multiple signaling pathways, involving nuclear erythroid 2-related factor 2 (Nrf2), may contribute to the redox balance, and thereby may be involved in glucose homeostasis. The aim of this study is to investigate whether SCFAs increase glucose uptake by upregulating the endogenous antioxidant glutathione (GSH) in C2C12 myotubes. METHODS: C2C12 myotubes were exposed to 1, 5, or 20 mM of single (acetate, propionate, or butyrate) or mixtures of SCFAs for 24 h. Cytotoxicity, glucose uptake, and intracellular GSH levels were measured. RESULTS: 20 mM of mixture but not separate SCFAs induced cytotoxicity. Exposure to a mixture of SCFAs at 5 mM increased glucose uptake in myotubes, while 20 mM of propionate, butyrate, and mixtures decreased glucose uptake. Exposure to single SCFAs increased GSH levels in myotubes; however, SCFAs did not prevent the menadione-induced decrease in glucose uptake in myotubes. CONCLUSIONS: The effect of SCFAs on modulating glucose uptake in myotubes is not associated with the effect on endogenous GSH levels.

Hesperidin Functions as an Ergogenic Aid by Increasing Endothelial Function and Decreasing Exercise-Induced Oxidative Stress and Inflammation, Thereby Contributing to Improved Exercise Performance.

The regulation of blood flow to peripheral muscles is crucial for proper skeletal muscle functioning and exercise performance. During exercise, increased mitochondrial oxidative phosphorylation leads to increased electron leakage and consequently induces an increase in ROS formation, contributing to DNA, lipid, and protein damage. Moreover, exercise may increase blood- and intramuscular inflammatory factors leading to a deterioration in endurance performance. The aim of this review is to investigate the potential mechanisms through which the polyphenol hesperidin could lead to enhanced exercise performance, namely improved endothelial function, reduced exercise-induced oxidative stress, and inflammation. We selected in vivo RCTs, animal studies, and in vitro studies in which hesperidin, its aglycone form hesperetin, hesperetin-metabolites, or orange juice are supplemented at any dosage and where the parameters related to endothelial function, oxidative stress, and/or inflammation have been measured. The results collected in this review show that hesperidin improves endothelial function (via increased NO availability), inhibits ROS production, decreases production and plasma levels of pro-inflammatory markers, and improves anaerobic exercise outcomes (e.g., power, speed, energy). For elite and recreational athletes, hesperidin could be used as an ergogenic aid to enhance muscle recovery between training sessions, optimize oxygen and nutrient supplies to the muscles, and improve anaerobic performance.

The response of three-dimensional pancreatic alpha and beta cell co-cultures to oxidative stress.

The pancreatic islets of Langerhans have low endogenous antioxidant levels and are thus especially sensitive to oxidative stress, which is known to influence cell survival and behaviour. As bioengineered islets are gaining interest for therapeutic purposes, it is important to understand how their composition can be optimized to diminish oxidative stress. We investigated how the ratio of the two main islet cell types (alpha and beta cells) and their culture in three-dimensional aggregates could protect against oxidative stress. Monolayer and aggregate cultures were established by seeding the alphaTC1 (alpha) and INS1E (beta) cell lines in varying ratios, and hydrogen peroxide was applied to induce oxidative stress. Viability, oxidative stress, and the level of the antioxidant glutathione were measured. Both aggregation and an increasing prevalence of INS1E cells in the co-cultures conferred greater resistance to cell death induced by oxidative stress. Increasing the prevalence of INS1E cells also decreased the number of alphaTC1 cells experiencing oxidative stress in the monolayer culture. In 3D aggregates, culturing the alphaTC1 and INS1E cells in a ratio of 50:50 prevented oxidative stress in both cell types. Together, the results of this study lead to new insight into how modulating the composition and dimensionality of a co-culture can influence the oxidative stress levels experienced by the cells.

Synthetic Materials that Affect the Extracellular Matrix via Cellular Metabolism and Responses to a Metabolic State.

In regenerative medicine and tissue engineering, many materials are developed to mimic the extracellular matrix (ECM). However, these ECM-mimicking materials do not yet completely recapitulate the diversity and complexity of biological tissue-specific ECM. In this review, an alternative strategy is proposed to generate ECM, namely synthesizing a material that functions as a drug delivery system, releasing molecules that target cellular metabolic pathways and thereby stimulate the local cells to create their own ECM. This is based on the fact that ECM synthesis, modification, composition, signaling, stiffness, and degradation are modulated by cellular metabolism. Metabolism can be targeted at different levels, ranging from modulating the availability of substrates or co-factors to regulating the activity of essential transcription factors. Depending on the drug of interest, its characteristics, mechanism of action, cellular target, and application, a different drug delivery system should be designed. Metabolic drugs modulating the ECM require cellular uptake for their function, therefore reversible linkers are recommended. Preferably the metabolic modulators are only released when needed, which will be upon a specific metabolic state, a change in ECM stiffness, or ECM remodeling. Therefore, reversible linkers that respond to an environmental stimulus could be incorporated. All in all, a novel strategy is suggested to develop a tissue-specific ECM by generating a synthetic material that releases metabolic molecules modulating the ECM. Various ways to modulate the ECM properties via the metabolism are reviewed and guidelines for the development of these materials are provided.

The Role of Pancreatic Alpha Cells and Endothelial Cells in the Reduction of Oxidative Stress in Pseudoislets.

Pancreatic beta cells have inadequate levels of antioxidant enzymes, and the damage induced by oxidative stress poses a challenge for their use in a therapy for patients with type 1 diabetes. It is known that the interaction of the pancreatic endocrine cells with support cells can improve their survival and lead to less vulnerability to oxidative stress. Here we investigated alpha (alpha TC-1), beta (INS1E) and endothelial (HUVEC) cells assembled into aggregates known as pseudoislets as a model of the pancreatic islets of Langerhans. We hypothesised that the coculture of alpha, beta and endothelial cells would be protective against oxidative stress. First, we showed that adding endothelial cells decreased the percentage of oxidative stress-positive cells. We then asked if the number of endothelial cells or the size (number of cells) of the pseudoislet could increase the protection against oxidative stress. However, no additional benefit was observed by those changes. On the other hand, we identified a potential supportive effect of the alpha cells in reducing oxidative stress in beta and endothelial cells. We were able to link this to the incretin glucagon-like peptide-1 (GLP-1) by showing that the absence of alpha cells in the pseudoislet caused increased oxidative stress, but the addition of GLP-1 could restore this. Together, these results provide important insights into the roles of alpha and endothelial cells in protecting against oxidative stress.

The Role of Alpha Cells in the Self-Assembly of Bioengineered Islets.

Vascularization is undoubtedly one of the greatest challenges in tissue engineering. Its importance is particularly evident when considering the transplantation of (bioengineered) pancreatic islets of Langerhans, which are highly sensitive to the delivery of oxygen and nutrients for their survival and function. Here we studied pseudoislets of Langerhans, which are three-dimensional spheroids composed of ß (INS1E), α (alpha TC-1), and endothelial (HUVEC) cells, and were interested in how the location and prevalence of the different cell types affected the presence of endothelial cells in the pseudoislet. We hypothesized that alpha (α) cells play an essential role in islet self-assembly and the incorporation of endothelial cells into the pseudoislet, and are thus important to consider in tissue engineering or regenerative medicine strategies, which typically focuses on the insulin-producing beta (ß) cells alone. We first determined the effect of changing the relative ratios of the cells and found the cell distribution converged on a steady state of ∼21% α cells, 74% ß cells, and 5% endothelial cells after 10 days of culture regardless of their respective ratios at seeding. We also found that the incorporation of endothelial cells was related to the pseudoislet size, with more endothelial cells found in the core of larger pseudoislets following a concomitant increase of α cells and a decrease in ß cells. Finally, we observed that both endothelial and ß cells were found adjacent to α cells significantly more frequently than to each other. In conclusion, this study demonstrates that the self-assembly of a pseudoislet is an intrinsically cell-regulated process. The endothelial cells had preferential proximity to the α cells, and this persisted even when challenged with changing the cell ratios and numbers. This study gives insight into the rules governing the self-organization of pseudoislets and suggests an important role for α cells to promote the incorporation of endothelial cells.

Increased Cell Survival of Human Primary Conjunctival Stem Cells in Dimethyl Sulfoxide-Based Cryopreservation Media.

Glycerol and dimethyl sulfoxide (DMSO) are widely used cryoprotectants for freezing human cell cultures. During the manufacturing process of ocular stem cell-based autographs, ex vivo cultivated ocular cells are cryopreserved for quality control purposes in accordance with regulatory requirements. The efficiency of the cryopreservation methods is limited by their effect on cell survival and quality. We compared two cryopreservation reagents, glycerol and DMSO, for their influence on the survival and quality of human primary conjunctival cultures. We found increased cell viability after cryopreservation in DMSO compared to cryopreservation in glycerol. The clonogenic and proliferative capacity was unaffected by the cryopreservation reagents, as shown by the colony forming efficiency and cumulative cell doubling. Importantly, the percentage of p63α- and keratin 19 (K19)-positive cells following cryopreservation in DMSO or glycerol was comparable. Taken together, our results demonstrate that cryopreservation in DMSO improves cell survival compared to cryopreservation in glycerol, with no subsequent effect on cell proliferative-, clonogenic-, or differentiation capacity. Therefore, we advise the use of a 10% DMSO-based cryopreservation medium for the cryopreservation of human primary conjunctival cells, as it will improve the number of cells available for the manufacturing of conjunctival stem cell-based autografts for clinical use.

Oxidative stress in pancreatic alpha and beta cells as a selection criterion for biocompatible biomaterials.

The clinical success rate of islet transplantation, namely independence from insulin injections, is limited by factors that lead to graft failure, including inflammation, acute ischemia, acute phase response, and insufficient vascularization. The ischemia and insufficient vascularization both lead to high levels of oxidative stress, which are further aggravated by islet encapsulation, inflammation, and undesirable cell-biomaterial interactions. To identify biomaterials that would not further increase damaging oxidative stress levels and that are also suitable for manufacturing a beta cell encapsulation device, we studied five clinically approved polymers for their effect on oxidative stress and islet (alpha and beta cell) function. We found that 300 poly(ethylene oxide terephthalate) 55/poly(butylene terephthalate) 45 (PEOT/PBT300) was more resistant to breakage and more elastic than other biomaterials, which is important for its immunoprotective function. In addition, it did not induce oxidative stress or reduce viability in the MIN6 beta cell line, and even promoted protective endogenous antioxidant expression over 7 days. Importantly, PEOT/PBT300 is one of the biomaterials we studied that did not interfere with insulin secretion in human islets.

Building Complex Life Through Self-Organization.

Cells are inherently conferred with the ability to self-organize into the tissues and organs comprising the human body. Self-organization can be recapitulated in vitro and recent advances in the organoid field are just one example of how we can generate small functioning elements of organs. Tissue engineers can benefit from the power of self-organization and should consider how they can harness and enhance the process with their constructs. For example, aggregates of stem cells and tissue-specific cells benefit from the input of carefully selected biomolecules to guide their differentiation toward a mature phenotype. This can be further enhanced by the use of technologies to provide a physiological microenvironment for self-organization, enhance the size of the constructs, and enable the long-term culture of self-organized structures. Of importance, conducting self-organization should be limited to fine-tuning and should avoid over-engineering that could counteract the power of inherent cellular self-organization. Impact Statement Self-organization is a powerful innate feature of cells that can be fine-tuned but not over-engineered to create new tissues and organs.

Redox regulation in regenerative medicine and tissue engineering: The paradox of oxygen.

One of the biggest challenges in tissue engineering and regenerative medicine is to incorporate a functioning vasculature to overcome the consequences of a lack of oxygen and nutrients in the tissue construct. Otherwise, decreased oxygen tension leads to incomplete metabolism and the formation of the so-called reactive oxygen species (ROS). Cells have many endogenous antioxidant systems to ensure a balance between ROS and antioxidants, but if this balance is disrupted by factors such as high levels of ROS due to long-term hypoxia, there will be tissue damage and dysfunction. Current attempts to solve the oxygen problem in the field rarely take into account the importance of the redox balance and are instead centred on releasing or generating oxygen. The first problem with this approach is that although oxygen is necessary for life, it is paradoxically also a highly toxic molecule. Furthermore, although some oxygen-generating biomaterials produce oxygen, they also generate hydrogen peroxide, a ROS, as an intermediate product. In this review, we discuss why it would be a superior strategy to supplement oxygen delivery with molecules to safeguard the important redox balance. Redox sensor proteins that can stimulate the anaerobic metabolism, angiogenesis, and enhancement of endogenous antioxidant systems are discussed as promising targets. We propose that redox regulating biomaterials have the potential to tackle some of the challenges related to angiogenesis and that the knowledge in this review will help scientists in tissue engineering and regenerative medicine realize this aim.

Activation versus inhibition of microsomal glutathione S-transferase activity by acrolein. Dependence on the concentration and time of acrolein exposure.

The toxicity of acrolein, an α,ß-unsaturated aldehyde, is due to its soft electrophilic nature and primarily involves the adduction of protein thiols. The thiol glutathione (GSH) forms the first line of defense against acrolein. The present study confirms that acrolein added to isolated rat liver microsomes can increase microsomal GSH transferase (MGST) activity 2-3 fold, which can be seen as a direct adaptive increase in the protection against acrolein. At a relatively high exposure level, acrolein appeared to inhibit MGST. The activation is due to adduction of thiol groups, and the inactivation probably involves adduction of amino groups in the enzyme by acrolein. The preference of acrolein to react with thiol groups over amino groups can explain why the enzyme is activated at a low exposure level and inhibited at a high exposure level of acrolein. These opposite forms of direct adaptation on the level of enzyme activity further narrow the thin line between survival and promotion of cell death, governed by the level of exposure.

Rutin protects against H2O2-triggered impaired relaxation of placental arterioles and induces Nrf2-mediated adaptation in Human Umbilical Vein Endothelial Cells exposed to oxidative stress.

BACKGROUND: Rutin intake is associated with a reduced risk of cardiovascular disease (CVD). The exact mechanism by which rutin can protect against CVD development is still enigmatic. Since, rutin is a compound with a relatively short half-life, the direct antioxidant effect of rutin cannot explain the long-lasting effect on human health. We hypothesized that rutin next to its direct antioxidant effect that improves endothelial function, may also induce an adaptive response in endogenous antioxidant systems. METHODS AND RESULTS: In Human Umbilical Vein Endothelial Cells (HUVECs), the direct antioxidant effect was confirmed. During scavenging of Reactive Oxygen Species (ROS), rutin is oxidized into a quinone derivative. HUVECs pretreated with rutin quinone became better protected against a second challenge with oxidative stress 3h later. LC-MS/MS analysis indicated that rutin quinone targets cysteine 151 of Keap1. Moreover, we found that the quinone is an inhibitor of the selenoprotein thioredoxin reductase 1. These properties correlated with an activation of Nrf2 and upregulation of Glutamate Cysteine Ligase, the rate-limiting enzyme of glutathione synthesis, while NF-κB and HIF activation became blunted by rutin treatment. Furthermore, rutin was found to prevent hydrogen peroxide from impairing relaxation of human chorionic plate placental vessels, which may help to protect endothelial function. CONCLUSION AND SIGNIFICANCE: Rutin functions as an antioxidant and is oxidized into a quinone that upregulates the Nrf2-mediated endogenous antioxidant response. This mechanism suggests that rutin selectively exerts its protective effects in regions with increased oxidative stress, and explains how rutin reduces the risk of developing CVD. GENERAL SIGNIFICANCE: The newly found mechanism behind the long-term protection of rutin against cardiovascular disease, the selective upregulation of endogenous antioxidant systems, contributes to the further understanding why rutin can reduce the risk on developing cardiovascular disease.

Silver nanoparticles induce hormesis in A549 human epithelial cells.

Despite the gaps in our knowledge on the toxicity of silver nanoparticles (AgNPs), the application of these materials is fast expanding, from medicine, to food as well as the use in consumer products. It has been reported that prolonged exposure might make cells more resistant to AgNPs. This prompted us to investigate if AgNPs may give rise to a hormetic response. Two types of AgNPs were used, i.e. colloidal AgNPs and an AgNP powder. For both types of nanosilver it was found that a low dose pretreatment of A549 human epithelial cells with AgNPs induced protection against a toxic dose of AgNPs and acrolein. This protection was more pronounced after pretreatment with the colloidal AgNPs. Interestingly, the mechanism of the hormetic response appeared to differ from that of acrolein. Adaptation to acrolein is related to Nrf2 translocation, increased mRNA expression of γGCS, HO-1 and increased GSH levels and the increased GSH levels can explain the hormetic effect. The adaptive response to AgNPs was not related to an increase in mRNA expression of γGCS and GSH levels. Yet, HO-1 mRNA expression and Nrf2 immunoreactivity were enhanced, indicating that these processes might be involved. So, AgNPs induce adaptation, but in contrast to acrolein GSH plays no role.

Time in Redox Adaptation Processes: From Evolution to Hormesis.

Life on Earth has to adapt to the ever changing environment. For example, due to introduction of oxygen in the atmosphere, an antioxidant network evolved to cope with the exposure to oxygen. The adaptive mechanisms of the antioxidant network, specifically the glutathione (GSH) system, are reviewed with a special focus on the time. The quickest adaptive response to oxidative stress is direct enzyme modification, increasing the GSH levels or activating the GSH-dependent protective enzymes. After several hours, a hormetic response is seen at the transcriptional level by up-regulating Nrf2-mediated expression of enzymes involved in GSH synthesis. In the long run, adaptations occur at the epigenetic and genomic level; for example, the ability to synthesize GSH by phototrophic bacteria. Apparently, in an adaptive hormetic response not only the dose or the compound, but also time, should be considered. This is essential for targeted interventions aimed to prevent diseases by successfully coping with changes in the environment e.g., oxidative stress.

Adaptation to acrolein through upregulating the protection by glutathione in human bronchial epithelial cells: the materialization of the hormesis concept.

Acrolein is a thiol reactive compound present in cigarette smoke and plays a pivotal role in the deleterious effects of smoking. Acrolein causes toxicity in human bronchial epithelial cells in a dose dependent manner. GSH forms the first line of defense against acrolein-induced toxicity. At high doses of acrolein (⩾10 µM) the capacity of the cellular protection by GSH is overwhelmed and GSH is not able to quench all the acrolein, resulting in cytotoxicity. At a relatively low dose of acrolein (3 µM), no cytotoxicity is observed due to protection by GSH. Moreover we found that exposure to a low dose of acrolein protects cells against the toxic effect of a second higher dose of acrolein. The adaptation to acrolein is induced via Nrf2 mediated gene expression of γ-glutamylcysteine synthetase leading to elevated GSH levels. This upregulation of the protection by GSH demonstrates a hormetic response to acrolein. Hormesis is an adaptive or compensatory response induced by a relatively subtle challenge of homeostasis by a toxic compound. Insight into the mechanism of hormesis is mandatory for a more accurate societal regulation of toxic compounds.