Spatial trans-epithelial electrical resistance (S-TEER) integrated in organs-on-chips

none6sìTransepithelial/transendothelial electrical resistance (TEER) is a label-free assay that is commonly used to assess tissue barrier integrity. TEER measurement systems have been embedded in organ-on-a-chip devices to provide live readouts of barrier functionality. Yet, these systems commonly provide the impedance values which correspond to the highest level of permeability throughout the chip and cannot provide localized information on specific regions of interest. This work introduces a system that provides this essential information: a spatial-TEER (S-TEER) organ-on-a-chip platform, which incorporates moving (scanning) electrodes that can measure electrical resistance at any desired location along the chip. We demonstrate the system's capacity to obtain localized measurements of permeability in selected regions of a cell sample. We show how, in a layer with non-uniform levels of cell coverage, permeability is higher in areas with lower cell density-suggesting that the system can be used to monitor local cellular growth in vitro. To demonstrate the applicability of the chip in studies of barrier function, we characterize tissue response to TNF-α and to EGTA, agents known to harm tissue barrier integrity.openNoa Renous , Mark D Kiri , Ronny A Barnea , Rossana Rauti , Yael Leichtmann-Bardoogo , Ben M MaozRenous, Noa; D Kiri, Mark; A Barnea, Ronny; Rauti, Rossana; Leichtmann-Bardoogo, Yael; M Maoz, Be

Ferritin is secreted via 2 distinct nonclassical vesicular pathways

Ferritin turnover plays a major role in tissue iron homeostasis, and ferritin malfunction is associated with impaired iron homeostasis and neurodegenerative diseases. In most eukaryotes, ferritin is considered an intracellular protein that stores iron in a nontoxic and bioavailable form. In insects, ferritin is a classically secreted protein and plays a major role in systemic iron distribution. Mammalian ferritin lacks the signal peptide for classical endoplasmic reticulum–Golgi secretion but is found in serum and is secreted via a nonclassical lysosomal secretion pathway. This study applied bioinformatics and biochemical tools, alongside a protein trafficking mouse models, to characterize the mechanisms of ferritin secretion. Ferritin trafficking via the classical secretion pathway was ruled out, and a 2:1 distribution of intracellular ferritin between membrane-bound compartments and the cytosol was observed, suggesting a role for ferritin in the vesicular compartments of the cell. Focusing on nonclassical secretion, we analyzed mouse models of impaired endolysosomal trafficking and found that ferritin secretion was decreased by a BLOC-1 mutation but increased by BLOC-2, BLOC-3, and Rab27A mutations of the cellular trafficking machinery, suggesting multiple export routes. A 13-amino-acid motif unique to ferritins that lack the secretion signal peptide was identified on the BC-loop of both subunits and plays a role in the regulation of ferritin secretion. Finally, we provide evidence that secretion of iron-rich ferritin was mediated via the multivesicular body–exosome pathway. These results enhance our understanding of the mechanism of ferritin secretion, which is an important piece in the puzzle of tissue iron homeostasis

Effect of SARS-CoV-2 proteins on vascular permeability.

Severe acute respiratory syndrome (SARS)-CoV-2 infection leads to severe disease associated with cytokine storm, vascular dysfunction, coagulation, and progressive lung damage. It affects several vital organs, seemingly through a pathological effect on endothelial cells. The SARS-CoV-2 genome encodes 29 proteins, whose contribution to the disease manifestations, and especially endothelial complications, is unknown. We cloned and expressed 26 of these proteins in human cells and characterized the endothelial response to overexpression of each, individually. Whereas most proteins induced significant changes in endothelial permeability, nsp2, nsp5_c145a (catalytic dead mutant of nsp5), and nsp7 also reduced CD31, and increased von Willebrand factor expression and IL-6, suggesting endothelial dysfunction. Using propagation-based analysis of a protein–protein interaction (PPI) network, we predicted the endothelial proteins affected by the viral proteins that potentially mediate these effects. We further applied our PPI model to identify the role of each SARS-CoV-2 protein in other tissues affected by coronavirus disease (COVID-19). While vali-dating the PPI network model, we found that the tight junction (TJ) proteins cadherin-5, ZO-1, and β-catenin are affected by nsp2, nsp5_c145a, and nsp7 consistent with the model prediction. Overall, this work identifies the SARS-CoV-2 proteins that might be most detrimental in terms of endothelial dysfunction, thereby shedding light on vascular aspects of COVID-1

Role of antioxidant enzymes in cardioprotective regimens of cold acclimation

The effect of cold on the organism depends on the degree of cold and its duration. Cold acclimation induces a thermoregulatory response, shivering thermogenesis and subsequently the development of non-shivering thermogenesis associated with the activation of brown adipose tissue. Exposure to mild cold in the form of hardening possesses beneficial effects on the whole organism and recently the cardioprotective effects of mild cold acclimation has been described. Increased antioxidant capacity has been demonstrated in the mechanism of cardioprotection induced by hypoxia. The aim of this thesis was to find out whether the protective effect of mild cold acclimatization (8 ± 1 řC) is associated with a change in the capacity of the antioxidant system. To achieve this goal, the protein levels of the main antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase, thioredoxin, thioredoxin reductase, aconitase, heme oxygenase) were determined during acclimation to mild cold, specifically in 1-3-10 days and subsequently after 5 weeks, and after a 2-week recovery. The results showed changes in most of the antioxidant proteins, however the effect on cold-elicited cardioprotection must be further verified. Key words: heart, cold acclimation, superoxide dismutase, catalase, glutathione...Účinek chladu na organismus závisí na míře chladu a době trvání. Chladová aklimace vyvolává termoregulační odpověď, třesovou termogenezi a následně rozvoj termogeneze netřesové spojené s aktivací hnědé tukové tkáně. Expozice mírnému chladu ve formě otužování má blahodárné účinky na celý organismus a nedávno bylo zjištěno, že má také kardioprotektivní účinky. Zvýšená antioxidační kapacita byla prokázána v mechanismu kardioprotekce vyvolané aklimací na hypoxii. Cílem této diplomové práce bylo zjistit, zda je protektivní efekt mírné chladové aklimace (8 ± 1 řC) spojen se změnou kapacity antioxidačního systému. V rámci tohoto cíle byly stanoveny hladiny proteinů hlavních antioxidačních enzymů (superoxiddismutáza, kataláza, glutathionperoxidáza, thioredoxin, thioredoxinreduktáza, akonitáza, hemoxygenáza) v průběhu aklimace na mírný chlad, konkrétně v 1-3-10 dnech, a následně po 5 týdnech a po 2-týdenním zotavení. Výsledky ukázaly změny v expresi většiny zkoumaných antioxidačních proteinů, jejichž vliv na kardioprotekci vyvolanou chladem musí být dále ověřen. Klíčová slova: srdce, chladová aklimace, superoxiddismutáza, kataláza, glutathionperoxidáza, thioredoxin, thioredoxinreduktáza, akonitáza, hemoxygenázaDepartment of PhysiologyKatedra fyziologieFaculty of SciencePřírodovědecká fakult

Estudio de las vías de señalización implicadas en la sobreexpresión de la proteína hemooxigenasa-1 en células endoteliales progenitoras estimuladas con secretoma aterosclerótico

Las enfermedades cardiovasculares siguen siendo una de las principales causas de muerte en el mundo. La etiología subyacente a estas enfermedades es la aterosclerosis, que conlleva un proceso inflamatorio exacerbado que afecta a la pared arterial. La aterosclerosis comienza con la activación las células endoteliales que recubren el interior de la arteria debido a diversos factores físico-químicos y mayoritariamente en respuesta a estrés oxidativo. Esto hace que el endotelio sea más permeable al paso de ciertas sustancias como lipoproteínas de baja densidad (LDL) que se oxida en su interior (LDLox) y a ciertos tipos celulares como los macrófagos, que fagocitan las LDLox convirtiéndose en células espumosas, que pierden su movilidad y que liberan citoquinas quimioatrayentes que reclutan más células a la zona de lesión. Con el avance de la enfermedad, entran en juego la necrosis o la calcificación, pudiendo producirse la ruptura de la placa obstruyendo el vaso, o formando un trombo que cause necrosis en otras zonas periféricas. En la actualidad no ha hay ningún tratamiento que consiga remitir el proceso aterosclerótico y devolver el tejido a su estado inicial, únicamente tratamientos farmacológicos que frenan el avance de la lesión, así como cirugías de revascularización, amputaciones, etc. Existen diversas terapias en estudio que implican el uso de activadores farmacológicos de proteínas antiinflamatorias y antioxidantes como HMOX1. Es de especial interés el uso de terapias celulares que implicarían el uso de células endoteliales progenitoras (EPC) del propio paciente. En trabajos previos del grupo, se estudió la expresión diferencial de EPCs en respuesta a la incubación directa con los secretomas procedentes de arterias ateroscleróticas frente a arterias no-ateroscleróticas, encontrando variaciones de ciertas proteínas, entre ellas la sobreexpresión de HMOX1. De acuerdo con estos resultados, en el presente trabajo de Tesis se ha analizado más en profundidad el efecto de factores ateroscleróticos sobre los niveles de HMOX1, así como otras proteínas relacionadas en EPCs, dado su potencial interés como agente antiaterosclerótico. Para ello se ha estudiado la ruta de señalización de HMOX1 desde un doble enfoque, proteómico y génico. En ambos enfoques, se han encontrado cambios en la expresión y la regulación de las proteínas y genes implicados en la ruta de señalización dependientes del tiempo y las concentraciones de incubación

Eisenstoffwechsel im Maushoden : die Bedeutung von IRP2 (Iron Regulatory Protein 2) für die testikuläre Eisenhomöostase in der Maus

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Orchestrated regulation of iron trafficking proteins in the kidney during iron overload facilitates systemic iron retention.

The exact route of iron through the kidney and its regulation during iron overload are not completely elucidated. Under physiologic conditions, non-transferrin and transferrin bound iron passes the glomerular filter and is reabsorbed through kidney epithelial cells, so that hardly any iron is found in the urine. To study the route of iron reabsorption through the kidney, we analyzed the location and regulation of iron metabolism related proteins in kidneys of mice with iron overload, elicited by iron dextran injections. Transferrin Receptor 1 was decreased as expected, following iron overload. In contrast, the multi-ligand hetero-dimeric receptor-complex megalin/cubilin, which also mediates the internalization of transferrin, was highly up-regulated. Moreover, with increasing iron, intracellular ferritin distribution shifted in renal epithelium from an apical location to a punctate distribution throughout the epithelial cells. In addition, in contrast to many other tissues, the iron exporter ferroportin was not reduced by iron overload in the kidney. Iron accumulated mainly in interstitial macrophages, and more prominently in the medulla than in the cortex. This suggests that despite the reduction of Transferrin Receptor 1, alternative pathways may effectively mediate re-absorption of iron that cycles through the kidney during parenterally induced iron-overload. The most iron consuming process of the body, erythropoiesis, is regulated by the renal erythropoietin producing cells in kidney interstitium. We propose, that the efficient re-absorption of iron by the kidney, also during iron overload enables these cells to sense systemic iron and regulate its usage based on the systemic iron state