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.

MiRNAs as Potential Regulators of Enthesis Healing: Findings in a Rodent Injury Model.

MicroRNAs (miRNAs) are short non-coding RNA sequences with the ability to inhibit the expression of a target mRNA at the post-transcriptional level, acting as modulators of both the degenerative and regenerative processes. Therefore, these molecules constitute a potential source of novel therapeutic tools. In this study, we investigated the miRNA expression profile that presented in enthesis tissue upon injury. For this, a rodent enthesis injury model was developed by creating a defect at a rat's patellar enthesis. Following injury, explants were collected on days 1 (n = 10) and 10 (n = 10). Contra lateral samples (n = 10) were harvested to be used for normalization. The expression of miRNAs was investigated using a "Fibrosis" pathway-focused miScript qPCR array. Later, target prediction for the aberrantly expressed miRNAs was performed by means of the Ingenuity Pathway Analysis, and the expression of mRNA targets relevant for enthesis healing was confirmed using qPCRs. Additionally, the protein expression levels of collagens I, II, III, and X were investigated using Western blotting. The mRNA expression pattern of EGR1, COL2A1, RUNX2, SMAD1, and SMAD3 in the injured samples indicated their possible regulation by their respective targeting miRNA, which included miR-16, -17, -100, -124, -133a, -155 and -182. Furthermore, the protein levels of collagens I and II were reduced directly after the injury (i.e., day 1) and increased 10 days post-injury, while collagens III and X showed the opposite pattern of expression.

Enhanced Microvasculature Formation and Patterning in iPSC-Derived Kidney Organoids Cultured in Physiological Hypoxia.

Stem cell-derived kidney organoids have been shown to self-organize from induced pluripotent stem cells into most important renal structures. However, the structures remain immature in culture and contain endothelial networks with low connectivity and limited organoid invasion. Furthermore, the nephrons lose their phenotype after approximately 25 days. To become applicable for future transplantation, further maturation in vitro is essential. Since kidneys in vivo develop in hypoxia, we studied the modulation of oxygen availability in culture. We hypothesized that introducing long-term culture at physiological hypoxia, rather than the normally applied non-physiological, hyperoxic 21% O2, could initiate angiogenesis, lead to enhanced growth factor expression and improve the endothelial patterning. We therefore cultured the kidney organoids at 7% O2 instead of 21% O2 for up to 25 days and evaluated nephrogenesis, growth factor expression such as VEGF-A and vascularization. Whole mount imaging revealed a homogenous morphology of the endothelial network with enhanced sprouting and interconnectivity when the kidney organoids were cultured in hypoxia. Three-dimensional vessel quantification confirmed that the hypoxic culture led to an increased average vessel length, likely due to the observed upregulation of VEGFA-189 and VEGFA-121, and downregulation of the antiangiogenic protein VEGF-A165b measured in hypoxia. This research indicates the importance of optimization of oxygen availability in organoid systems and the potential of hypoxic culture conditions in improving the vascularization of organoids.

AMPK Modulates the Metabolic Adaptation of C6 Glioma Cells in Glucose-Deprived Conditions without Affecting Glutamate Transport.

Energy homeostasis in the central nervous system largely depends on astrocytes, which provide metabolic support and protection to neurons. Astrocytes also ensure the clearance of extracellular glutamate through high-affinity transporters, which indirectly consume ATP. Considering the role of the AMP-activated protein kinase (AMPK) in the control of cell metabolism, we have examined its implication in the adaptation of astrocyte functions in response to a metabolic stress triggered by glucose deprivation. We genetically modified the astrocyte-like C6 cell line to silence AMPK activity by overexpressing a dominant negative mutant of its catalytic subunit. Upon glucose deprivation, we found that C6 cells maintain stable ATP levels and glutamate uptake capacity, highlighting their resilience during metabolic stress. In the same conditions, cells with silenced AMPK activity showed a reduction in motility, metabolic activity, and ATP levels, indicating that their adaptation to stress is compromised. The rate of ATP production remained, however, unchanged by AMPK silencing, suggesting that AMPK mostly influences energy consumption during stress conditions in these cells. Neither AMPK modulation nor prolonged glucose deprivation impaired glutamate uptake. Together, these results indicate that AMPK contributes to the adaptation of astrocyte metabolism triggered by metabolic stress, but not to the regulation of glutamate transport.

Oxidative Stress-Induced Sirtuin1 Downregulation Correlates to HIF-1α, GLUT-1, and VEGF-A Upregulation in Th1 Autoimmune Hashimoto's Thyroiditis.

In Hashimoto's thyroiditis (HT), oxidative stress (OS) is driven by Th1 cytokines' response interfering with the normal function of thyrocytes. OS results from an imbalance between an excessive production of reactive oxygen species (ROS) and a lowering of antioxidant production. Moreover, OS has been shown to inhibit Sirtuin 1 (SIRT1), which is able to prevent hypoxia-inducible factor (HIF)-1α stabilization. The aims of this study were to determine the involvement of NADPH-oxidases (NOX), SIRT1, and HIF-1α in HT pathophysiology as well as the status of antioxidant proteins such as peroxiredoxin 1 (PRDX1), catalase, and superoxide dismutase 1 (SOD1). The protein expressions of NOX2, NOX4, antioxidant enzymes, SIRT1, and HIF-1α, as well as glucose transporter-1 (GLUT-1) and vascular endothelial growth factor A (VEGF-A), were analyzed by Western blot in primary cultures of human thyrocytes that were or were not incubated with Th1 cytokines. The same proteins were also analyzed by immunohistochemistry in thyroid samples from control and HT patients. In human thyrocytes incubated with Th1 cytokines, NOX4 expression was increased whereas antioxidants, such as PRDX1, catalase, and SOD1, were reduced. Th1 cytokines also induced a significant decrease of SIRT1 protein expression associated with an upregulation of HIF-1α, GLUT-1, and VEGF-A proteins. With the exception of PRDX1 and SOD1, similar results were obtained in HT thyroids. OS due to an increase of ROS produced by NOX4 and a loss of antioxidant defenses (PRDX1, catalase, SOD1) correlates to a reduction of SIRT1 and an upregulation of HIF 1α, GLUT-1, and VEGF-A. Our study placed SIRT1 as a key regulator of OS and we, therefore, believe it could be considered as a potential therapeutic target in HT.

miR-199a Downregulation as a Driver of the NOX4/HIF-1α/VEGF-A Pathway in Thyroid and Orbital Adipose Tissues from Graves' Patients.

Graves' disease (GD) is an autoimmune thyroiditis often associated with Graves' orbitopathy (GO). GD thyroid and GO orbital fat share high oxidative stress (OS) and hypervascularization. We investigated the metabolic pathways leading to OS and angiogenesis, aiming to further decipher the link between local and systemic GD manifestations. Plasma and thyroid samples were obtained from patients operated on for multinodular goiters (controls) or GD. Orbital fats were from GO or control patients. The NADPH-oxidase-4 (NOX4)/HIF-1α/VEGF-A signaling pathway was investigated by Western blotting and immunostaining. miR-199a family expression was evaluated following quantitative real-time PCR and/or in situ hybridization. In GD thyroids and GO orbital fats, NOX4 was upregulated and correlated with HIF-1α stabilization and VEGF-A overexpression. The biotin assay identified NOX4, HIF-1α and VEGF-A as direct targets of miR-199a-5p in cultured thyrocytes. Interestingly, GD thyroids, GD plasmas and GO orbital fats showed a downregulation of miR-199a-3p/-5p. Our results also highlighted an activation of STAT-3 signaling in GD thyroids and GO orbital fats, a transcription factor known to negatively regulate miR-199a expression. We identified NOX4/HIF-1α/VEGF-A as critical actors in GD and GO. STAT-3-dependent regulation of miR-199a is proposed as a common driver leading to these events in GD thyroids and GO orbital fats.

Downregulation of Caveolin-1 and Upregulation of Deiodinase 3, Associated with Hypoxia-Inducible Factor-1α Increase, Are Involved in the Oxidative Stress of Graves' Orbital Adipocytes.

Background: Even though the clinical features of Graves' orbitopathy (GO) are well known, its exact pathogenesis remains controversial. The imbalance of redox homeostasis in the connective tissue could play a crucial role leading to an inflammatory state and edema of soft orbital tissues, thus contributing to orbital hypoxia and increase in hypoxia-inducible factor (HIF)-1α. This oxidative stress appears to target the orbital cells such as fibroblasts and also adipocytes. This study aims to explore which pathways can lead to the aforementioned oxidative stress in GO adipose cells and therefore offers new plausible therapeutic targets. Methods: Orbital fat samples were obtained from patients with GO (Western blot [WB]: n = 8, immunohistochemistry [IHC]: n = 8) and from control patients (WB: n = 5, IHC: n = 3-5). They were processed for WB analysis and IHC of the antioxidants (catalase, superoxide dismutase 1) and for HIF-1α. The expression of caveolin-1 (Cav-1) and deiodinase 3 (DIO3), known to be regulated by HIF-1α, was also analyzed by WB and IHC, as well as the targets of Cav-1: glucose transporter type 4 (Glut-4), NADPH oxidase (NOX)-2, and endothelial nitric oxide synthase (eNOS). Triiodothyronine (T3) expression was also analyzed by IHC. Results: In GO adipocytes, the expression of catalase was reduced, whereas that of HIF-1α was strongly increased. A decreased local T3 supply was associated with DIO3 upregulation. The low expression of Cav-1 in GO adipocytes was associated not only with low expression of Glut-4 but also with an increased expression of NOX-2 and active eNOS phosphorylated on serine 1177. Conclusions: Cav-1 and DIO3, both sensitive to hypoxia and to the increase of HIF-1α, play a pivotal role in the oxidative stress in GO adipocytes. DIO3 regulates the cellular supply of T3, which is essential for the cell homeostasis. Cav-1 determines the cellular glucose supply through Glut-4 and regulates the activity of NOX-2 generating superoxide anions and that of eNOS generating nitric oxide (NO).

Brief O2 uploading during continuous hypothermic machine perfusion is simple yet effective oxygenation method to improve initial kidney function in a porcine autotransplant model.

With oxygenation proposed as a resuscitative measure during hypothermic models of preservation, the aim of this study was to evaluate the optimal start time of oxygenation during continuous hypothermic machine perfusion (HMP). In this porcine ischemia-reperfusion autotransplant model, the left kidney of a ±40 kg pig was exposed to 30 minutes of warm ischemia prior to 22 hours of HMP and autotransplantation. Kidneys were randomized to receive 2 hours of oxygenation during HMP either at the start (n = 6), or end of the perfusion (n = 5) and outcomes were compared to standard, nonoxygenated HMP (n = 6) and continuous oxygenated HMP (n = 8). The brief initial and continuous oxygenated HMP groups were associated with superior graft recovery compared to either standard, nonoxygenated HMP or kidneys oxygenated at the end of HMP. This correlated with significant metabolic differences in perfusate (eg, lactate, succinate, flavin mononucleotide) and tissues (eg, succinate, adenosine triphosphate, hypoxia-inducible factor-1α, nuclear factor erythroid 2-related factor 2) suggesting superior mitochondrial preservation with initial oxygenation. Brief initial O2 uploading during HMP at procurement site might be an easy and effective preservation strategy to maintain aerobic metabolism, protect mitochondria, and achieve an improved early renal graft function compared with standard HMP or oxygen supply shortly at the end of HMP preservation.

Influence of Different Partial Pressures of Oxygen During Continuous Hypothermic Machine Perfusion in a Pig Kidney Ischemia-reperfusion Autotransplant Model.

BACKGROUND: The optimal perfusate partial pressure of oxygen (PO2) during hypothermic machine perfusion (HMP) is unknown. The aims of the study were to determine the functional, metabolic, structural, and flow dynamic effects of low and high perfusate PO2 during continuous HMP in a pig kidney ischemia-reperfusion autotransplant model. METHODS: The left kidneys of a ±40 kg pigs were exposed to 30 minutes of warm ischemia and randomized to receive 22-hour HMP with either low perfusate PO2 (30% oxygen, low oxygenated HMP [HMPO2]) (n = 8) or high perfusate PO2 (90% oxygen, HMPO2high) (n = 8), before autotransplantation. Kidneys stored in 22-hour standard HMP (n = 6) and 22-hour static cold storage (n = 6) conditions served as controls. The follow-up after autotransplantation was 13 days. RESULTS: High PO2 resulted in a 3- and 10-fold increase in perfusate PO2 compared with low HMPO2 and standard HMP, respectively. Both HMPO2 groups were associated with superior graft recovery compared with the control groups. Oxygenation was associated with a more rapid and sustained decrease in renal resistance. While there was no difference in functional outcomes between both HMPO2 groups, there were clear metabolic differences with an inverse correlation between oxygen provision and the concentration of major central metabolites in the perfusion fluid but no differences were observed by oxidative stress and metabolic evaluation on preimplantation biopsies. CONCLUSIONS: While this animal study does not demonstrate any advantages for early graft function for high perfusate PO2, compared with low perfusate PO2, perfusate metabolic profile analysis suggests that aerobic mechanism is better supported under high perfusate PO2 conditions.

MicroRNA-199a-3p and MicroRNA-199a-5p Take Part to a Redundant Network of Regulation of the NOS (NO Synthase)/NO Pathway in the Endothelium.

Objective- Members of the microRNA (miR)-199a family, namely miR-199a-5p and miR-199a-3p, have been recently identified as potential regulators of cardiac homeostasis. Also, upregulation of miR-199a expression in cardiomyocytes was reported to influence endothelial cells. Whether miR-199a is expressed by endothelial cells and, if so, whether it directly regulates endothelial function remains unknown. We investigate the implication of miR-199a products on endothelial function by focusing on the NOS (nitric oxide synthase)/NO pathway. Approach and Results- Bovine aortic endothelial cells were transfected with specific miRNA inhibitors (locked-nucleic acids), and potential molecular targets identified with prediction algorithms were evaluated by Western blot or immunofluorescence. Ex vivo experiments were performed with mice treated with antagomiRs targeting miR-199a-3p or -5p. Isolated vessels and blood were used for electron paramagnetic resonance or myograph experiments. eNOS (endothelial NO synthase) activity (through phosphorylations Ser1177/Thr495) is increased by miR-199a-3p/-5p inhibition through an upregulation of the PI3K (phosphoinositide 3-kinase)/Akt (protein kinase B) and calcineurin pathways. SOD1 (superoxide dismutase 1) and PRDX1 (peroxiredoxin 1) upregulation was also observed in locked-nucleic acid-treated cells. Moreover, miR-199a-5p controls angiogenesis and VEGFA (vascular endothelial growth factor A) production and upregulation of NO-dependent relaxation were observed in vessels from antagomiR-treated mice. This was correlated with increased circulated hemoglobin-NO levels and decreased superoxide production. Angiotensin infusion for 2 weeks also revealed an upregulation of miR-199a-3p/-5p in vascular tissues. Conclusions- Our study reveals that miR-199a-3p and miR-199a-5p participate in a redundant network of regulation of the NOS/NO pathway in the endothelium. We highlighted that inhibition of miR-199a-3p and -5p independently increases NO bioavailability by promoting eNOS activity and reducing its degradation, thereby supporting VEGF-induced endothelial tubulogenesis and modulating vessel contractile tone.

Perfusion-decellularization of human ear grafts enables ECM-based scaffolds for auricular vascularized composite tissue engineering.

INTRODUCTION: Human ear reconstruction is recognized as the emblematic enterprise in tissue engineering. Up to now, it has failed to reach human applications requiring appropriate tissue complexity along with an accessible vascular tree. We hereby propose a new method to process human auricles in order to provide a poorly immunogenic, complex and vascularized ear graft scaffold. METHODS: 12 human ears with their vascular pedicles were procured. Perfusion-decellularization was applied using a SDS/polar solvent protocol. Cell and antigen removal was examined by histology and DNA was quantified. Preservation of the extracellular matrix (ECM) was assessed by conventional and 3D-histology, proteins and cytokines quantifications. Biocompatibility was assessed by implantation in rats for up to 60 days. Adipose-derived stem cells seeding was conducted on scaffold samples and with human aortic endothelial cells whole graft seeding in a perfusion-bioreactor. RESULTS: Histology confirmed cell and antigen clearance. DNA reduction was 97.3%. ECM structure and composition were preserved. Implanted scaffolds were tolerated in vivo, with acceptable inflammation, remodeling, and anti-donor antibody formation. Seeding experiments demonstrated cell engraftment and viability. CONCLUSIONS: Vascularized and complex auricular scaffolds can be obtained from human source to provide a platform for further functional auricular tissue engineered constructs, hence providing an ideal road to the vascularized composite tissue engineering approach. STATEMENT OF SIGNIFICANCE: The ear is emblematic in the biofabrication of tissues and organs. Current regenerative medicine strategies, with matrix from donor tissues or 3D-printed, didn't reach any application for reconstruction, because critically missing a vascular tree for perfusion and transplantation. We previously described the production of vascularized and cell-compatible scaffolds, from porcine ear grafts. In this study, we ---- applied findings directly to human auricles harvested from postmortem donors, providing a perfusable matrix that retains the ear's original complexity and hosts new viable cells after seeding. This approach unlocks the ability to achieve an auricular tissue engineering approach, associated with possible clinical translation.

Bioengineering a Human Face Graft: The Matrix of Identity.

OBJECTIVE: During the last decade, face allotransplantation has been shown to be a revolutionary reconstructive procedure for severe disfigurements. However, offer to patients remains limited due to lifelong immunosuppression. To move forward in the field, a new pathway in tissue engineering is proposed. BACKGROUND: Our previously reported technique of matrix production of a porcine auricular subunit graft has been translated to a human face model. METHODS: 5 partial and 1 total face grafts were procured from human fresh cadavers. After arterial cannulation, the specimens were perfused using a combined detergent/polar solvent decellularization protocol. Preservation of vascular patency was assessed by imaging, cell and antigen removal by DNA quantification and histology. The main extracellular matrix proteins and associated cytokines were evaluated. Lip scaffolds were cultivated with dermal, muscle progenitor and endothelial cells, either on discs or in a bioreactor. RESULTS: Decellularization was successful in all facial grafts within 12 days revealing acellular scaffolds with full preservation of innate morphology. Imaging demonstrated a preservation of the entire vascular tree patency. Removal of cells and antigens was confirmed by reduction of DNA and antigen markers negativation. Microscopic evaluation revealed preservation of tissue structures as well as of major proteins. Seeded cells were viable and well distributed within all scaffolds. CONCLUSIONS: Complex acellular facial scaffolds were obtained, preserving simultaneously a cell-friendly extracellular matrix and a perfusable vascular tree. This step will enable further engineering of postmortem facial grafts, thereby offering new perspectives in composite tissue allotransplantation.

Pioglitazone, a PPARγ Agonist, Upregulates the Expression of Caveolin-1 and Catalase, Essential for Thyroid Cell Homeostasis: A Clue to the Pathogenesis of Hashimoto's Thyroiditis.

BACKGROUND: Peroxisome proliferator-activated receptor γ (PPARγ) is a transcription factor that regulates the expression of multiple target genes involved in several metabolic pathways as well as in inflammation. The expression and cell localization of caveolin-1 (Cav-1), thyroperoxidase (TPO), and dual oxidase (DUOX), involved in extracellular iodination, is modulated by Th1 cytokines in human normal thyroid cells and in Hashimoto's thyroiditis (HT). OBJECTIVES: The objectives of this study were (i) to analyze the PPARγ protein and mRNA expression at the follicular level in HT versus controls in correlation with the one of Cav-1; (ii) to study the effects of Th1 cytokines on PPARγ and catalase expression in human thyrocyte primary cultures; and (iii) to study the effects of pioglitazone, a PPARγ agonist, on thyroxisome components (Cav-1, TPO, DUOX) and on catalase, involved in antioxidant defense. RESULTS: Although the global expression of PPARγ in the whole gland of patients with HT was not modified compared with controls, there was great heterogeneity among glands and among follicles within the same thyroid. Besides normal (type 1) follicles, there were around inflammatory zones, hyperactive (type 2) follicles with high PPARγ and Cav-1 expression, and inactive (type 3) follicles which were unable to form thyroxine and did not express PPARγ or Cav-1. In human thyrocytes in primary culture, Th1 cytokines decreased PPARγ and catalase expression; pioglitazone increased Cav-1, TPO, and catalase expression. CONCLUSION: PPARγ may play a central role in normal thyroid physiology by upregulating Cav-1, essential for the organization of the thyroxisome and extracellular iodination. By upregulating catalase, PPARγ may also contribute to cell homeostasis. The inhibitory effect of Th1 cytokines on PPARγ expression may be considered as a new pathogenetic mechanism for HT, and the use of PPARγ agonists could open a new therapeutic approach.