scholarly journals Nucleotide metabolic mismatches in mammalian hearts: implications for transplantation

2013 ◽  
Vol 95 (1) ◽  
pp. 9-14 ◽  
Author(s):  
Z Khalpey ◽  
MH Yacoub ◽  
RT Smolenski
Keyword(s):  
Endothelial Cells ◽  
Human Blood ◽  
Ex Vivo ◽  
Nucleotide Metabolism ◽  
Donor Organ ◽  
Human Donor ◽  
Genetic Modifications ◽  
Significant Difference

Introduction Human donor organ shortages have led surgeons and scientists to explore the use of animals as alternative organ sources. Acute thrombovascular rejection (AVR) is the main hurdle in xenotransplantation. Disparities in nucleotide metabolism in the vessels of different species may contribute significantly to the microvascular component of AVR. Methods We evaluated the extent of nucleotide metabolism mismatch in selected organs and endothelial cells of different mammals with particular focus on the changes in activity of ecto-5’-nucleotidase (E5’N) elicited by exposure of porcine hearts or endothelial cells to human blood (ex vivo) or human plasma (in vitro). Results E5’N activity in the rat heart was significantly higher than in other species. We noted a significant difference (p<0.001) in E5’N activity between human and pig endothelial cell lines. Initial pig aortic endothelial E5’N activity decreased in vitro after a three-hour exposure to human and porcine plasma while remaining constant in controls. Ex vivo perfusion with fresh human blood for four hours resulted in a significant decrease of E5’N activity in both wild type and transgenic pig hearts overexpressing human decay accelerating factor (p<0.001). Conclusions This study provides evidence that mismatches in basal mammalian metabolic pathways and humoral immunity interact in a xenogeneic environment. Understanding the role of nucleotide metabolism and signalling in xenotransplantation may identify new targets for genetic modifications and may lead to the development of new therapies extending graft survival.

scholarly journals A tumor-promoting role for soluble TβRIII in glioblastoma

2021 ◽  
Author(s):  
Isabel Burghardt ◽  
Judith Johanna Schroeder ◽  
Tobias Weiss ◽  
Dorothee Gramatzki ◽  
Michael Weller
Keyword(s):  
Endothelial Cells ◽  
Ex Vivo ◽  
Microvascular Endothelial Cells ◽  
Smad2 Phosphorylation ◽  
Cell Gene Expression ◽  
Microvascular Endothelial ◽  
Cell Gene ◽  
Mouse Glioma

Abstract Purpose Members of the transforming growth factor (TGF)-β superfamily play a key role in the regulation of the malignant phenotype of glioblastoma by promoting invasiveness, angiogenesis, immunosuppression, and maintaining stem cell-like properties. Betaglycan, a TGF-β coreceptor also known as TGF-β receptor III (TβRIII), interacts with members of the TGF-β superfamily and acts as membrane-associated or shed molecule. Shed, soluble TβRIII (sTβRIII) is produced upon ectodomain cleavage of the membrane-bound form. Elucidating the role of TβRIII may improve our understanding of TGF-β pathway activity in glioblastoma Methods Protein levels of TβRIII were determined by immunohistochemical analyses and ex vivo single-cell gene expression profiling of glioblastoma tissue respectively. In vitro, TβRIII levels were assessed investigating long-term glioma cell lines (LTCs), cultured human brain-derived microvascular endothelial cells (hCMECs), glioblastoma-derived microvascular endothelial cells, and glioma-initiating cell lines (GICs). The impact of TβRIII on TGF-β signaling was investigated, and results were validated in a xenograft mouse glioma model Results Immunohistochemistry and ex vivo single-cell gene expression profiling of glioblastoma tissue showed that TβRIII was expressed in the tumor tissue, predominantly in the vascular compartment. We confirmed this pattern of TβRIII expression in vitro. Specifically, we detected sTβRIII in glioblastoma-derived microvascular endothelial cells. STβRIII facilitated TGF-β-induced Smad2 phosphorylation in vitro and overexpression of sTβRIII in a xenograft mouse glioma model led to increased levels of Smad2 phosphorylation, increased tumor volume, and decreased survival Conclusions These data shed light on the potential tumor-promoting role of extracellular shed TβRIII which may be released by glioblastoma endothelium with high sTβRIII levels.

scholarly journals Metformin-stimulated AMPK-α1 promotes microvascular repair in acute lung injury

2013 ◽  
Vol 305 (11) ◽  
pp. L844-L855 ◽  
Author(s):  
Ming-Yuan Jian ◽  
Mikhail F. Alexeyev ◽  
Paul E. Wolkowicz ◽  
Jaroslaw W. Zmijewski ◽  
Judy R. Creighton
Keyword(s):  
Endothelial Cells ◽  
Acute Lung Injury ◽  
Lung Injury ◽  
Ex Vivo ◽  
Endothelial Permeability ◽  
Beneficial Effects ◽  
Isolated Lung

Acute lung injury secondary to sepsis is a leading cause of mortality in sepsis-related death. Present therapies are not effective in reversing endothelial cell dysfunction, which plays a key role in increased vascular permeability and compromised lung function. AMP-activated protein kinase (AMPK) is a molecular sensor important for detection and mediation of cellular adaptations to vascular disruptive stimuli. In this study, we sought to determine the role of AMPK in resolving increased endothelial permeability in the sepsis-injured lung. AMPK function was determined in vivo using a rat model of endotoxin-induced lung injury, ex vivo using the isolated lung, and in vitro using cultured rat pulmonary microvascular endothelial cells (PMVECs). AMPK stimulation using N1-(α-d-ribofuranosyl)-5-aminoimidizole-4-carboxamide or metformin decreased the LPS-induced increase in permeability, as determined by filtration coefficient ( Kf) measurements, and resolved edema as indicated by decreased wet-to-dry ratios. The role of AMPK in the endothelial response to LPS was determined by shRNA designed to decrease expression of the AMPK-α1 isoform in capillary endothelial cells. Permeability, wounding, and barrier resistance assays using PMVECs identified AMPK-α1 as the molecule responsible for the beneficial effects of AMPK in the lung. Our findings provide novel evidence for AMPK-α1 as a vascular repair mechanism important in the pulmonary response to sepsis and identify a role for metformin treatment in the management of capillary injury.

scholarly journals Endothelial cell-derived extracellular vesicles exert cardio-protective effect via their protein cargo

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
F Ravera ◽  
S Femmino' ◽  
C Penna ◽  
L Franchin ◽  
F Angelini ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Guanylyl Cyclase ◽  
Ex Vivo ◽  
Biological Effects ◽  
Transwell Assay ◽  
Rat Hearts ◽  
Cardio Protection

Abstract Background Extracellular vesicles (EV) are recognized as carriers of relevant biological effects and have been identified as regulators of cell-to-cell communication contributing to several patho-physiological processes. These processes include angiogenesis/coagulation/tissue repair/inflammation. In ischemia/reperfusion (I/R) settings, along with the direct effects of the I/R itself, paracrine mechanisms associated with the activation of the inflammatory response, primary involving endothelial cells, are crucial drivers of both vessel and cardiomyocyte damage. Purpose Since in models of myocardial I/R injury the role of EV released from endothelial cells is still unclear, our hypothesis was to provide insight on this specific topic. To this end, naïve endothelial cell (EC)-derived EV (eEV) and eEV released in response to the pro-inflammatory cytokine interleukin-3 (IL-3) (eEV-IL-3) have been evaluated on different I/R models. Methods eEV were characterized by MACSPlex-Exosome-Kit and western blot analysis. For the in-vitro hypoxia-reoxygenation (H/R) experiments, H9c2 or EC were pretreated with eEV, eEV-IL-3 (1x104 EV/cell) or IL-3 (10ng/ml) for 2 hours and then exposed to hypoxia (1% O2, 5% CO2) for additional 2 hours in the presence of eEV, eEV-IL-3 or IL-3 and subsequently reoxygenated (21% O2 and 5% CO2) for 1 hour. To verify the effect of EC treated with eEV, eEV-IL-3 or IL-3 on H9c2 and subjected to H/R protocol, transwell assay was used. At the end of the H/R protocol, cell viability was assessed. For ex-vivo experiments, isolated rat hearts, pretreated with a buffer containing EV (from EC pretreated or not with IL-3), were subjected to 30 minutes global normothermic ischemia and 1 hour reperfusion. Triton infusion was also used as a model of endothelial damage. At the end of I/R, the infarct size was measured and expressed as a percentage of total left ventricular mass (LVM). The role of eNOS/guanylyl-cyclase/MEK1/2 pathways in mediating eEV biological effects was also evaluated using different inhibitors both in in-vitro and ex-vivo models. Finally, protein profiles of eEV and eEV-IL-3 were analyzed using label free mass spectrometry. Results eEV and eEV-IL-3 protect EC, but not H9c2 exposed to H/R protocol, while eEV, but not eEV-IL-3-treatment limits I/R injury in the rat heart. Rat hearts pre-treated with triton significantly avoid eEV-induced cardio-protection. Transwell assay showed a reduction of H9C2 mortality after treatment with both eEV and eEV-IL-3. Proteomic analysis revealed that MEK1/2 and the endothelial-NOS (eNOS)-antagonist caveolin-1 were differentially expressed in eEV and eEV-IL-3. The use of eNOS/guanylyl-cyclase/MEK1/2 inhibitors prevented eEV-induced cardio-protection. Conclusions These observations indicate that eEV, but not eEV-IL-3, have cardio-protective effects when given as preconditioning agents. We have also shown that the activation of eNOS/GC/MEK1/2 pathway is crucial for eEV-mediated cardio-protection. Funding Acknowledgement Type of funding source: None

Modulatory role of human blood eosinophils in angiogenesis: Ex vivo and in vitro studies

2003 ◽  
Vol 111 (2) ◽  
pp. S211 ◽  
Author(s):  
I. Puxeddu ◽  
A. Alian ◽  
A.M. Piliponsky ◽  
A. Panet ◽  
F. Levi-Schaffer
Keyword(s):  
Human Blood ◽  
Ex Vivo ◽  
In Vitro Studies ◽  
Blood Eosinophils

The Role of Polyphenols in the Modulation of Plasma Non-Enzymatic Antioxidant Capacity (NEAC)

2012 ◽  
Vol 82 (3) ◽  
pp. 228-232 ◽  
Author(s):  
Mauro Serafini ◽  
Giuseppa Morabito
Keyword(s):  
Antioxidant Capacity ◽  
Dietary Intervention ◽  
Ex Vivo ◽  
The Body ◽  
Dietary Polyphenols ◽  
Enzymatic Antioxidant ◽  
Endogenous Antioxidant

Dietary polyphenols have been shown to scavenge free radicals, modulating cellular redox transcription factors in different in vitro and ex vivo models. Dietary intervention studies have shown that consumption of plant foods modulates plasma Non-Enzymatic Antioxidant Capacity (NEAC), a biomarker of the endogenous antioxidant network, in human subjects. However, the identification of the molecules responsible for this effect are yet to be obtained and evidences of an antioxidant in vivo action of polyphenols are conflicting. There is a clear discrepancy between polyphenols (PP) concentration in body fluids and the extent of increase of plasma NEAC. The low degree of absorption and the extensive metabolism of PP within the body have raised questions about their contribution to the endogenous antioxidant network. This work will discuss the role of polyphenols from galenic preparation, food extracts, and selected dietary sources as modulators of plasma NEAC in humans.

Antiangiogenic effects of phospholipase A2 Lys49 BnSP-7 from Bothrops pauloensis snake venom on endothelial cells: An in vitro and ex vivo approach

2021 ◽  
Vol 72 ◽  
pp. 105099
Author(s):  
Lorena Polloni ◽  
Fernanda Van Petten Vasconcelos Azevedo ◽  
Samuel Cota Teixeira ◽  
Eloá Moura ◽  
Tassia Rafaela Costa ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Phospholipase A2 ◽  
Snake Venom ◽  
Ex Vivo ◽  
Ex Vivo Approach

scholarly journals Neutrophil extracellular traps induce MCP-1 release from endothelial cells at the plaque rupture site in acute myocardial infarction

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
A Ondracek ◽  
T.M Hofbauer ◽  
A Mangold ◽  
T Scherz ◽  
V Seidl ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Acute Myocardial Infarction ◽  
Endothelial Cells ◽  
Plaque Rupture ◽  
Ex Vivo ◽  
Coronary Intervention ◽  
Neutrophil Extracellular Traps ◽  
Funding Source ◽  
Extracellular Traps

Abstract Introduction Leukocyte-mediated inflammation is crucial in acute myocardial infarction (AMI). We recently observed that neutrophil extracellular traps (NETs) are increased at the culprit site, promoting activation and differentiation of fibrocytes, cells with mesenchymal and leukocytic properties. Fibrocyte migration is mediated by monocyte chemoattractant protein (MCP)-1 and C-C chemokine receptor type 2 (CCR2). We investigated the interplay between NETs, fibrocyte function, and MCP-1 in AMI. Methods Culprit site and femoral blood of AMI patients was drawn during percutaneous coronary intervention. We characterized CCR2 expression of fibrocytes by flow cytometry. MCP-1 and the NET marker citrullinated histone H3 (citH3) were measured by ELISA. Fibrocytes were treated in vitro with MCP-1. Human coronary arterial endothelial cells (hCAECs) were stimulated with isolated NETs, and MCP-1 was measured by ELISA and qPCR. The influence of MCP-1 on NET formation in vitro was assessed using isolated neutrophils. Results We have included 50 consecutive AMI patients into the study. NETs and concentrations of MCP-1 were increased at the CLS. NET stimulation of hCAECs induced MCP-1 on mRNA and protein level. Increasing MCP-1 gradient was associated with fibrocyte accumulation at the site of occlusion. In the presence of higher MCP-1 these fibrocytes expressed proportionally less CCR2 than peripheral fibrocytes. In vitro, MCP-1 dose-dependently decreased fibrocyte CCR2 and reduced ex vivo NET release of healthy donor neutrophils. Conclusions NETs induce endothelial MCP-1 release, presumably promoting a chemotactic gradient for leukocyte and fibrocyte migration. MCP-1 mediated inhibition of NET formation could point to a negative feedback loop. These data will shed light on vascular healing. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Austrian Science Fund

scholarly journals Antagonism of Adherent Invasive E. coli LF82 With Human α-defensin 5 in the Follicle-associated Epithelium of Patients With Ileal Crohn’s Disease

2020 ◽  
Author(s):  
Lina Y Alkaissi ◽  
Martin E Winberg ◽  
Stéphanie DS Heil ◽  
Staffan Haapaniemi ◽  
Pär Myrelid ◽  
...  
Keyword(s):  
Crohn’S Disease ◽  
Crohn's Disease ◽  
Ex Vivo ◽  
Ussing Chambers ◽  
E Coli ◽  
Follicle Associated Epithelium ◽  
Vitro Model ◽  
Set Up

Abstract Background The first visible signs of Crohn’s disease (CD) are microscopic erosions over the follicle-associated epithelium (FAE). The aim of the study was to investigate the effects of human α-defensin 5 (HD5) on adherent-invasive Escherichia coli LF82 translocation and HD5 secretion after LF82 exposure in an in vitro model of human FAE and in human FAE ex vivo. Methods An in vitro FAE-model was set up by the coculture of Raji B cells and Caco-2-cl1 cells. Ileal FAE from patients with CD and controls were mounted in Ussing chambers. The effect of HD5 on LF82 translocation was studied by LF82 exposure to the cells or tissues with or without incubation with HD5. The HD5 secretion was measured in human FAE exposed to LF82 or Salmonella typhimurium. The HD5 levels were evaluated by immunofluorescence, immunoblotting, and ELISA. Results There was an increased LF82 translocation across the FAE-model compared with Caco-2-cl1 (P &lt; 0.05). Incubation of cell/tissues with HD5 before LF82 exposure reduced bacterial passage in both models. Human FAE showed increased LF82 translocation in CD compared with controls and attenuated passage after incubation with sublethal HD5 in both CD and controls (P &lt; 0.05). LF82 exposure resulted in a lower HD5 secretion in CD FAE compared with controls (P &lt; 0.05), whereas Salmonella exposure caused equal secretion on CD and controls. There were significantly lower HD5 levels in CD tissues compared with controls. Conclusions Sublethal HD5 reduces the ability of LF82 to translocate through FAE. The HD5 is secreted less in CD in response to LF82, despite a normal response to Salmonella. This further implicates the integrated role of antimicrobial factors and barrier function in CD pathogenesis.

scholarly journals A decellularized human corneal scaffold for anterior corneal surface reconstruction

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Naresh Polisetti ◽  
Anke Schmid ◽  
Ursula Schlötzer-Schrehardt ◽  
Philip Maier ◽  
Stefan J. Lang ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Ex Vivo ◽  
Sodium Deoxycholate ◽  
Nuclear Material ◽  
Biological Properties ◽  
Host Cells ◽  
Human Cornea ◽  
Human Donor ◽  
Short Period

AbstractAllogenic transplants of the cornea are prone to rejection, especially in repetitive transplantation and in scarred or highly vascularized recipient sites. Patients with these ailments would particularly benefit from the possibility to use non-immunogenic decellularized tissue scaffolds for transplantation, which may be repopulated by host cells in situ or in vitro. So, the aim of this study was to develop a fast and efficient decellularization method for creating a human corneal extracellular matrix scaffold suitable for repopulation with human cells from the corneal limbus. To decellularize human donor corneas, sodium deoxycholate, deoxyribonuclease I, and dextran were assessed to remove cells and nuclei and to control tissue swelling, respectively. We evaluated the decellularization effects on the ultrastructure, optical, mechanical, and biological properties of the human cornea. Scaffold recellularization was studied using primary human limbal epithelial cells, stromal cells, and melanocytes in vitro and a lamellar transplantation approach ex vivo. Our data strongly suggest that this approach allowed the effective removal of cellular and nuclear material in a very short period of time while preserving extracellular matrix proteins, glycosaminoglycans, tissue structure, and optical transmission properties. In vitro recellularization demonstrated good biocompatibility of the decellularized human cornea and ex vivo transplantation revealed complete epithelialization and stromal repopulation from the host tissue. Thus, the generated decellularized human corneal scaffold could be a promising biological material for anterior corneal reconstruction in the treatment of corneal defects.

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