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Induced pluripotent stem cell (iPSC)–derived kidney organoids have the potential for a large variety of applications. However, they do not persist long in culture, for which reasons are still unclear. Furthermore, their morphological maturation, an essential feature for kidney function, has not been sufficiently assessed. Kidney organoids are transcriptionally much alike end-of-first-trimester fetal kidneys and present many of the same cell types. From large transmission electron microscopy tilescans of specific regions of interest, we compared the ultrastructures of iPSC-derived kidney organoids at various timepoints to human fetal kidneys of the first trimester. Unlike healthy fetal kidneys, large glycogen deposits developed over time in all organoid cell types, but particularly in podocytes and in chondrocytes, one of the off-target populations that contaminate the culture. Deeper investigation showed that glycogen synthase kinase 3b (GSK3β) levels and activation were diminished over time, correlated with the accumulation of glycogen. Activated YAP was strongly expressed and large lipid droplets accumulated over time in proximal tubules. Accordingly, EGFR signaling increased significantly over time. Mitochondria in glomeruli and tubules contained few or no cristae, indicating mitochondrial damage. Together these features are known for hyperglycemic cultures and diabetic nephropathy. Measuring the glucose concentration in the kidney organoid culture medium showed a concentration of 2.94 g/mL, which is considered an elevated, pre-diabetic–like concentrationin vitro. In summary, our ultrastructural assessment of iPSC-derived kidney organoids using an age-matched fetal human reference allowed the evaluation of cellular morphology, and we identified intracellular features that can inform the cellular state, which is particularly important while physiological testing of organoids is limited.Translational StatementKidney organoids hold promise as a future treatment for patients with end-stage kidney disease. The engineering of kidney organoids with correct and healthy morphologyin vitrois therefore essential, to guarantee functionality after transplantation. The present study provided deeper insights into the structural organization and ultrastructure of cells in kidney organoids compared to age-matched human fetal kidneys. Accordingly, we found several features in the regular kidney organoid culture, which are known to occur in hyperglycemic cultures and diabetic nephropathy, indicating that the current medium composition may be inducing pathological cellular phenotypes. This study therefore creates a better understanding of current limitations in the kidney organoid culture, increases knowledge of their function and cellular organization, and sets the foundation for further research to create advanced organoids.

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.

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).

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.

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 O

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.

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.

Iodine deficiency (ID) induces TSH-independent microvascular activation in the thyroid via the reactive oxygen species/nitric oxide-hypoxia-inducible factor-1α/vascular endothelial growth factor (VEGF) pathway. We hypothesized the additional involvement of mammalian target of rapamycin (mTOR) as a positive regulator of this pathway and AMP-activated protein kinase (AMPK) as a negative feedback regulator to explain the transient nature of ID-induced microvascular changes under nonmalignant conditions. mTOR and AMPK involvement was investigated using an in vitro model (human thyrocytes in primary cultures) and 2 murine models of goitrogenesis (normal NMRI and RET-PTC mice [a papillary thyroid cancer model]). In NMRI mice, ID had no effect on the phosphorylation of ribosomal S6 kinase (p70S6K), a downstream target of mTOR. However, rapamycin inhibited ID-induced thyroid blood flow and VEGF protein expression. In the RET-PTC model, ID strongly increased the phosphorylation of p70S6K, whereas rapamycin completely inhibited the ID-induced increase in p70S6K phosphorylation, thyroid blood flow, and VEGF-A expression. In vitro, although ID increased p70S6K phosphorylation, the ID-stimulated hypoxia-inducible factor/VEGF pathway was inhibited by rapamycin. Activation of AMPK by metformin inhibited ID effects both in vivo and in vitro. In AMPK-α1 knockout mice, the ID-induced increase in thyroid blood flow and VEGF-A protein expression persisted throughout the treatment, whereas both parameters returned to control values in wild-type mice after 4 days of ID. In conclusion, mTOR is required for early ID-induced thyroid microvascular activation. AMPK negatively regulates this pathway, which may account for the transient nature of ID-induced TSH-independent vascular effects under benign conditions.

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.

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.

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).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.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.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.

Iodine deficiency (ID) induces microvascular changes in the thyroid gland via a TSH-independent reactive oxygen species-hypoxia inducible factor (HIF)-1α-vascular endothelial growth factor (VEGF) pathway. The involvement of nitric oxide (NO) in this pathway and the role of calcium (Ca(2+)) and of ryanodine receptors (RYRs) in NO synthase 3 (NOS3) activation were investigated in a murine model of goitrogenesis and in three in vitro models of ID including primary cultures of human thyrocytes. ID activated NOS3 and the production of NO in thyrocytes in vitro and increased the thyroid blood flow in vivo. Using bevacizumab (a blocking antibody against VEGF-A) in mice, it appeared that NOS3 is activated upstream of VEGF-A. L-nitro arginine methyl ester (L-NAME, a NOS inhibitor) blocked the ID-induced increase in thyroid blood flow in vivo and NO production in vitro, as well as ID-induced VEGF-A mRNA and HIF-1α expression in vitro, while S-nitroso-acetyl-penicillamine (SNAP, a NO donor) did the opposite. Ca(2+) is involved in this pathway as intracellular Ca(2+) flux increased after ID, and thapsigargin activated NOS3 and increased VEGF-A mRNA expression. Two of the three known mammalian RYR isoforms (RYR1 and RYR2) were shown to be expressed in thyrocytes. RYR inhibition using ryanodine at 10 μ M decreased ID-induced NOS3 activation, HIF-1α and VEGF-A expression, while RYR activation with ryanodine at 1nM increased NOS3 activation and VEGF-A mRNA expression. In conclusion, during the early phase of TSH-independent ID-induced microvascular activation, ID sequentially activates RYRs and NOS3, thereby supporting ID-induced activation of the NO/HIF-1α/VEGF-A pathway in thyrocytes.

458 Implication of microRNA 199a3p and 199a5p in vascular function : modulation of the eNOS/NO pathway {#article-title-2} Introduction: MiR 199a3p and 199a5p have been mainly implicated in proliferation, cell survival and remodeling in context of cancerogenesis but more recent data proposed a role in cardiovascular functions. Objectives: We seek to assess how miR199a3p and miR199a5p modulate endothelial function by identifying their molecular targets in endothelial cells. Methods: Mice were treated with miR199a3p/5p inhibitors and after 30 days, the contractile profile and endothelial function were evaluated ex vivo in the aorta. Cultured endothelial cells were transfected with miR199a3p/5p inhibitors or a scramble sequence. NO production, expression and/or activity of eNOS and its major regulators were measured by EPR and Western blot respectively. Implication of Akt pathway was analysed using LY294002 treatment. Angiogenesis was investigated in 2D-culture in matrigel support. Results: Vessels from mice treated with antagomirs against miR199a3p or 5p showed a larger NO-dependent relaxation compared to controls. Circulating HbNO measured by EPR in venous blood collected from these mice was significantly increased compared to controls suggesting a role of both miRs in the control of the NOS/ NO pathway. Repression of miR 199a3p/5p improved eNOS activity through an increased eNOS phosphorylation at serine 1177 and a decreased phosphorylation status of threonine 495. The eNOS allosteric regulator, cav-1, was not modulated by LNA treatments. However, both treatments promoted Akt activation and calcineurin expression. Furthermore, addition of LY294002 inhibited the increase of eNOS phosphorylation at serine 1177 induced by LNA treatment. Interestingly, repression of miR199a5p also promoted SOD expression, suggesting that miR199a5p also improved NO bioavailability. In addition, inhibition of miR199a5p upregulated VEGF production and promoted tubes formation in endothelial cells. Conclusion: Our results demonstrate that miR199a3p/5p modulate the NOS/NO pathway in the endothelium by promoting NO production and repressing NO degradation. These results also suggest a strong implication of the Akt pathway in this modulation. Interestingly, miR199a5p shows additional control of angiogenesis. # 459 Role of endothelial cell adenosine deaminase acting on RNA-2 in ischemic/inflammatory disease in vivo {#article-title-3} Background: RNA binding proteins (RBPs) are key players in posttranscriptional regulation of mRNAs. Adenosine deaminase acting on RNA-2 (ADAR2) enzyme binds to double-stranded RNAs and controls brain development. The role of ADAR2 in endothelial cell function has not been described so far. Methods and Results: ADAR2 is expressed in human and murine endothelial cells and is 2-fold induced by hypoxia or hind limb ischemia in mice (P