Study of human cornea endothelial cells migration into iatrogenic defect zone in experiment ex vivo

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

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A decellularized human corneal scaffold for anterior corneal surface reconstruction

Scientific Reports ◽

10.1038/s41598-021-82678-3 ◽

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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Can Be miR-126-3p a Biomarker of Premature Aging? An Ex Vivo and In Vitro Study in Fabry Disease

Cells ◽

10.3390/cells10020356 ◽

2021 ◽

Vol 10 (2) ◽

pp. 356 ◽

Cited By ~ 1

Author(s):

Alessia Lo Curto ◽

Simona Taverna ◽

Maria Assunta Costa ◽

Rosa Passantino ◽

Giuseppa Augello ◽

...

Keyword(s):

Endothelial Cells ◽

Fabry Disease ◽

Healthy Subjects ◽

Aging Process ◽

Replicative Senescence ◽

Ex Vivo ◽

Umbilical Vein ◽

Quantitative Polymerase Chain Reaction ◽

Premature Aging

Fabry disease (FD) is a lysosomal storage disorder (LSD) characterized by lysosomal accumulation of glycosphingolipids in a wide variety of cytotypes, including endothelial cells (ECs). FD patients experience a significantly reduced life expectancy compared to the general population; therefore, the association with a premature aging process would be plausible. To assess this hypothesis, miR-126-3p, a senescence-associated microRNA (SA-miRNAs), was considered as an aging biomarker. The levels of miR-126-3p contained in small extracellular vesicles (sEVs), with about 130 nm of diameter, were measured in FD patients and healthy subjects divided into age classes, in vitro, in human umbilical vein endothelial cells (HUVECs) “young” and undergoing replicative senescence, through a quantitative polymerase chain reaction (qPCR) approach. We confirmed that, in vivo, circulating miR-126 levels physiologically increase with age. In vitro, miR-126 augments in HUVECs underwent replicative senescence. We observed that FD patients are characterized by higher miR-126-3p levels in sEVs, compared to age-matched healthy subjects. We also explored, in vitro, the effect on ECs of glycosphingolipids that are typically accumulated in FD patients. We observed that FD storage substances induced in HUVECs premature senescence and increased of miR-126-3p levels. This study reinforces the hypothesis that FD may aggravate the normal aging process.

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A tumor-promoting role for soluble TβRIII in glioblastoma

Molecular and Cellular Biochemistry ◽

10.1007/s11010-021-04128-y ◽

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.

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Profiling of Cytokines Secreted by Conventional Aqueous Outflow Pathway Endothelial Cells Activated In Vitro and Ex Vivo With Laser Irradiation

Investigative Opthalmology & Visual Science ◽

10.1167/iovs.15-17660 ◽

2015 ◽

Vol 56 (12) ◽

pp. 7100 ◽

Cited By ~ 3

Author(s):

Jorge A. Alvarado ◽

Phuonglan Chau ◽

Jianfeng Wu ◽

Richard Juster ◽

Amde Selassie Shifera ◽

...

Keyword(s):

Endothelial Cells ◽

Laser Irradiation ◽

Ex Vivo ◽

Outflow Pathway ◽

Aqueous Outflow

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C5a and C5aR1 are key drivers of microvascular platelet aggregation in clinical entities spanning from aHUS to COVID-19

Blood Advances ◽

10.1182/bloodadvances.2021005246 ◽

2021 ◽

Author(s):

Sistiana Aiello ◽

Sara Gastoldi ◽

Miriam Galbusera ◽

Piero Luigi Ruggenenti ◽

Valentina Portalupi ◽

...

Keyword(s):

Endothelial Cells ◽

Bacterial Infections ◽

Viral Infections ◽

Ex Vivo ◽

Inflammatory Diseases ◽

Thrombus Formation ◽

Alternative Pathway ◽

Atypical Hemolytic Uremic Syndrome ◽

Uremic Syndrome

Unrestrained activation of the complement system till the terminal products, C5a and C5b-9, plays a pathogenetic role in acute and chronic inflammatory diseases. In endothelial cells, complement hyperactivation may translate into cell dysfunction, favoring thrombus formation. The aim of this study was to investigate the role of the C5a/C5aR1 axis as opposite to C5b-9 in inducing endothelial dysfunction and loss of anti-thrombogenic properties. In vitro and ex vivo assays with serum from patients with atypical hemolytic uremic syndrome (aHUS) -a prototype rare disease of complement-mediated microvascular thrombosis due to genetically determined alternative pathway dysregulation- and cultured microvascular endothelial cells, demonstrated that the C5a/C5aR1 axis is a key player of endothelial thromboresistance loss. C5a added to normal human serum, fully recapitulated the pro-thrombotic effects of aHUS serum. Mechanistic studies showed that C5a caused RalA-mediated exocytosis of vWF and P-selectin from Weibel-Palade bodies, which favored further vWF binding on the endothelium and platelet adhesion and aggregation. In patients with severe COVID-19 -who suffered from acute activation of complement triggered by SARS-CoV-2 infection- we found the same C5a-dependent pathogenic mechanisms. These results highlight C5a/C5aR1 as a common pro-thrombogenic effector spanning from genetic rare diseases to viral infections, and may have clinical implications. Selective C5a/C5aR1 blockade could have advantages over C5 inhibition, since the former preserves the formation of C5b-9 that is critical to control bacterial infections that often develop as comorbidities in severely ill patients. (Clinicaltrials.gov identifier NCT02464891)

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STUDY OF THE EFFECT OF HYPOTHERMIC CONSERVATION ON THE INTRACELLULAR SODIUM CONCENTRATION IN THE ENDOTHELIUM OF CORNEAL TRANSPLANTS

Vavilov Journal of Genetics and Breeding ◽

10.18699/vj18.379 ◽

2018 ◽

Vol 22 (4) ◽

pp. 433-437

Author(s):

G. S. Baturina ◽

I. G. Palchikova ◽

A. A. Konev ◽

E. S. Smirnov ◽

L. E. Katkova ◽

...

Keyword(s):

Endothelial Cells ◽

Plasma Membranes ◽

Sodium Concentration ◽

Intracellular Sodium ◽

Sodium Balance ◽

Human Cornea ◽

Sodium Permeability ◽

Custom Made ◽

Intracellular Sodium Concentration ◽

Cornea Endothelium

Endothelial keratoplasty has become the treatment of choice for corneal endothelial dysfunction. Advancements in the surgical treatment of corneal endothelial diseases depend on progress in graft conservation and its related advantages in assessing the suitability of grafts for transplantation. Transport of water and ions by cornea endothelium is important for the optic properties of cornea. In this work, we study the intracellular sodium concentration in cornea endothelial cells in samples of pig cornea that underwent hypothermic conservation for 1 and 10 days and endothelial cells of human cornea grafts after 10-day conservation. The concentration of intracellular sodium in preparations of endothelial cells was assayed using fluorescent dye SodiumGreen. The fluorescent images were analyzed with the custom-made computer program CytoDynamics. An increased level of intracellular sodium was shown in the endothelium after 10-day conservation in comparison with one-day conservation (pig samples). Sodium permeability of pig endothelial cell plasma membranes significantly decreased in these samples. Assessment of intracellular sodium in human cornea endothelium showed a higher level – as was in analogues pig samples of the corneal endothelium. The assay of the intracellular sodium balance concentration established in endothelial cells after hypothermic conservation in mediums L-15 and Optisol-GS showed a significant advantage of specialized me dium Optisol-GS. The balanced intracellular concentration after 10 days of hypothermic conservation was significantly lower in cells incubated at 4 °C in Optisol-GS (L-15, 128 ± 14, n = 15; Optisol-GS, 108 ± 14, n = 11; mM, p < 0.001). Intracellular sodium concentration could be a useful parameter for assessing cornea endothelium cell viability.

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My D88-Dependent Interplay Between Myeloid and Endothelial Cells in the Initiation and Progression of Obesity-Associated Inflammatory Diseases

Blood ◽

10.1182/blood.v128.22.sci-44.sci-44 ◽

2016 ◽

Vol 128 (22) ◽

pp. SCI-44-SCI-44

Author(s):

Xiaoxia Li

Keyword(s):

Insulin Resistance ◽

Endothelial Cells ◽

Adipose Tissue ◽

Systemic Inflammation ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Inflammatory Diseases ◽

Critical Role ◽

Low Grade

Abstract Low-grade systemic inflammation is often associated with metabolic syndrome, which plays a critical role in the development of the obesity-associated inflammatory diseases, including insulin resistance and atherosclerosis. Here, we investigate how Toll-like receptor-MyD88 signaling in myeloid and endothelial cells coordinately participates in the initiation and progression of high fat diet-induced systemic inflammation and metabolic inflammatory diseases. MyD88 deficiency in myeloid cells inhibits macrophage recruitment to adipose tissue and their switch to an M1-like phenotype. This is accompanied by substantially reduced diet-induced systemic inflammation, insulin resistance, and atherosclerosis. MyD88 deficiency in endothelial cells results in a moderate reduction in diet-induced adipose macrophage infiltration and M1 polarization, selective insulin sensitivity in adipose tissue, and amelioration of spontaneous atherosclerosis. Both in vivo and ex vivo studies suggest that MyD88-dependent GM-CSF production from the endothelial cells might play a critical role in the initiation of obesity-associated inflammation and development of atherosclerosis by priming the monocytes in the adipose and arterial tissues to differentiate into M1-like inflammatory macrophages. Collectively, these results implicate a critical MyD88-dependent interplay between myeloid and endothelial cells in the initiation and progression of obesity-associated inflammatory diseases. Disclosures No relevant conflicts of interest to declare.

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

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00173.2013 ◽

2013 ◽

Vol 305 (11) ◽

pp. L844-L855 ◽

Cited By ~ 47

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.

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