Shear flow increases S-nitrosylation of proteins in endothelial cells

During their passage through the circulatory system, tumor cells undergo extensive interactions with various host cells including endothelial cells. The capacity of tumor cells to form metastasis is related to their ability to interact with and extravasate through endothelial cell layers, which involves multiple adhesive interactions between tumor cells and endothelium (EC). Thus it is essential to identify the adhesive receptors on the endothelial and melanoma surface that mediate those specific adhesive interactions. P-selectin and E-selectin have been reported as adhesion molecules that mediate the cell-cell interaction of endothelial cells and melanoma cells. However, not all melanoma cells express ligands for selectins. In this study, we elucidated the molecular constituents involved in the endothelial adhesion and extravasation of sialyl-Lewisx/a-negative melanoma cell lines under flow in the presence and absence of polymorphonuclear neutrophils (PMNs). Results show the interactions of α4β1(VLA-4) on sialyl-Lewisx/a-negative melanoma cells and vascular adhesion molecule (VCAM-1) on inflamed EC supported melanoma adhesion to and subsequent extravasation through the EC in low shear flow. These findings provide clear evidence for a direct role of the VLA-4/VCAM-1 pathway in melanoma cell adhesion to and extravasation through the vascular endothelium in a shear flow. PMNs facilitated melanoma cell extravasation under both low and high shear conditions via the involvement of distinct molecular mechanisms. In the low shear regime, β2-integrins were sufficient to enhance melanoma cell extravasation, whereas in the high shear regime, selectin ligands and β2-integrins on PMNs were necessary for facilitating the melanoma extravasation process.

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Shear Flow Increases Insulin Sensitivity To Endothelial Cells Via S-Nitrosation of PTP1B

Free Radical Biology and Medicine ◽

10.1016/j.freeradbiomed.2011.10.234 ◽

2011 ◽

Vol 51 ◽

pp. S164

Author(s):

Ling D. Wang

Keyword(s):

Endothelial Cells ◽

Insulin Sensitivity ◽

Shear Flow

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Shear flow-driven actin re-organization induces ICAM-1 nanoclustering on endothelial cells that impact T-cell migration

10.1101/2020.06.29.177816 ◽

2020 ◽

Author(s):

Izabela K. Piechocka ◽

Sarah Keary ◽

Alberto Sosa-Costa ◽

Lukas Lau ◽

Nitin Mohan ◽

...

Keyword(s):

T Cells ◽

Endothelial Cells ◽

Cell Migration ◽

T Cell ◽

Shear Flow ◽

Membrane Receptors ◽

Mechanical Forces ◽

Shear Forces ◽

T Cell Migration ◽

The Impact

ABSTRACTThe leukocyte specific β2-integrin LFA-1, and its ligand ICAM-1 expressed on endothelial cells (ECs), are involved in the arrest, adhesion and transendothelial migration of leukocytes. Although the role of mechanical forces on LFA-1 activation is well established, the impact of forces on its major ligand ICAM-1, has received less attention. Using a parallel-plate flow chamber combined with confocal and super-resolution microscopy, we show that prolonged shear-flow induces a global translocation of ICAM-1 on ECs upstream of flow direction. Interestingly, shear-forces promoted ICAM-1 nanoclustering prior to LFA-1 engagement. This spatial nanoscale organization was driven by actin cytoskeleton re-arrangements induced by shear-force. We further assessed the impact of prolonged shear-stress EC stimulation on T cell migration. T cells adhered to mechanically pre-stimulated ECs developed a more pro-migratory phenotype, migrated faster and exhibited shorter EC interactions than when adhered to non-mechanically stimulated ECs. Together, our results indicate that shear-forces increase the number of ICAM-1/LFA-1 bonds due to ICAM-1 nanoclustering, strengthening adhesion and thereby reducing actin retrograde flow of T-cells, leading to their increased migration speed. Our data also underscores the importance of mechanical forces regulating the spatial organization of cell membrane receptors and their contribution to adhesion regulation, regardless of integrin activation.Summary statementWe show that shear forces promote ICAM-1 spatial re-arrangement and actin-dependent nanoclustering on ECs prior to integrin engagement. This mechanism might be important for firm leukocyte adhesion and migration during the immune response.

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Transmission of Anaplasma phagocytophilum from endothelial cells to peripheral granulocytes in vitro under shear flow conditions

Medical Microbiology and Immunology ◽

10.1007/s00430-015-0387-0 ◽

2015 ◽

Vol 204 (5) ◽

pp. 593-603 ◽

Cited By ~ 3

Author(s):

Jinyong Wang ◽

Viktor Dyachenko ◽

Ulrike G. Munderloh ◽

Reinhard K. Straubinger

Keyword(s):

Endothelial Cells ◽

Shear Flow ◽

Anaplasma Phagocytophilum ◽

Flow Conditions

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Inhibition of Hydrophobic Protein-MediatedCandida albicans Attachment to Endothelial Cells during Physiologic Shear Flow

Infection and Immunity ◽

10.1128/iai.69.5.2815-2820.2001 ◽

2001 ◽

Vol 69 (5) ◽

pp. 2815-2820 ◽

Cited By ~ 33

Author(s):

Pati M. Glee ◽

Jim E. Cutler ◽

Evelyn E. Benson ◽

Robert F. Bargatze ◽

Kevin C. Hazen

Keyword(s):

Endothelial Cells ◽

Shear Flow ◽

Cell Surface ◽

Protective Efficacy ◽

Cell Surface Hydrophobicity ◽

Surface Hydrophobicity ◽

Host Cells ◽

Yeast Cells ◽

Cytokine Activation ◽

Endothelial Monolayers

ABSTRACT Adhesion interactions during hematogenous dissemination ofCandida albicans likely involve a complex array of host and fungal factors. Possible C. albicans factors include changes in cell surface hydrophobicity and exposed antigens that have been shown in static adhesion assays to influence attachment events. We used a novel in vitro shear analysis system to investigate host-pathogen interactions and the role of fungal cell surface hydrophobicity in adhesion events with human endothelial cells under simulated physiologic shear. Endothelial monolayers were grown in capillary tubes and tested with and without interleukin-1β activation in buffered medium containing human serum. Hydrophobic and hydrophilic stationary-phase C. albicans yeast cells were infused into the system under shear flow and found to adhere with widely varying efficiencies. The average number of adherent foci was determined from multiple fields, sampled via video microscopy, between 8 and 12 min after infusion. Hydrophobic C. albicans cells demonstrated significantly more heterotypic binding events (Candida-endothelial cell) and greater homotypic binding events (Candida-Candida) than hydrophilic yeast cells. Cytokine activation of the endothelium significantly increased binding by hydrophobic C. albicans compared to unactivated host cells. Preincubation of hydrophobic yeast cells with a monoclonal antibody against hydrophobic cell wall proteins significantly blocked adhesion interactions with the endothelial monolayers. Because the antibody also blocks C. albicans binding to laminin and fibronectin, results suggest that vascular adhesion events with endothelial cells and exposed extracellular matrix may be blocked during C. albicans dissemination. Future studies will address the protective efficacy of blocking or redirecting blood-borne fungal cells to favor host defense mechanisms.

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Interrogating the Interaction of CD52 Functionalised Metallic Nanoparticles with Malignant B Lymphocytes

Blood ◽

10.1182/blood.v126.23.4437.4437 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4437-4437

Author(s):

Laura Christine Kickham ◽

Anthony M. McElligott ◽

Adriele Prina-Mello ◽

Elisabeth A. Vandenberghe ◽

Yuri Volkov ◽

...

Keyword(s):

Endothelial Cells ◽

Shear Flow ◽

B Lymphocytes ◽

Flow Cytometric Analysis ◽

Ph Sensitive ◽

Fe2o3 Nanoparticles ◽

Proof Of Concept ◽

Fluid Shear ◽

Flow Cytometric ◽

Cytotoxicity Studies

Abstract Chronic Lymphocytic Leukaemia (CLL) is a common B-lymphoid malignancy with over 200,000 people affected annually in Europe and the US. The aim of therapy is to increase the quality and duration of life using well tolerated treatment. Novel intracellular drug delivery systems such as functionalised nanoparticles (NPs), conjugated to antibodies such as anti-CD20 or anti-CD52 directed at cell surface markers may help address this need. We have explored the feasibility of targeting nanoparticles in CLL using a microfluidics based adhesion assay, anti CD52 cell targeting and Fludarabine therapy. Methods B cells were isolated from the peripheral blood of normal healthy donors and CLL patients. The CLL cell line, I-83, was maintained under standard conditions. Epifluorescent, laser scanning confocal and electron microscopy was utilised for imaging the interaction of metallic NPs with cells. The metallic NPs were polymer-coated for biocompatibility and cellular toxicity was assessed using flow cytometric analysis based on changes in light scattering. NPs distribution on the surface of the cells was visualized using epifluorescent and Helium ion microscopy, cellular uptake and alterations in cell morphology after NP treatment was imaged by confocal microscopy. Cell adhesion and migration behaviour under fluid shear flow conditions mimicking CLL cells in vivo was investigated using a microfluidics system utilising biochips coated with VCAM-1 and seeded with Human Umbilical Vein Endothelial Cells (HUVEC) or Human Dermal Lymphatic Endothelial Cells. CD52-Alexa Fluor® 633 was conjugated to the surface of silanized NPs (NP1) using standard carbo-diimide cross linker chemistry techniques and successful functionalisation of NPs was validated using flow cytometric analysis, monitoring a shift in fluorescent population. I-83 cells and patient-derived malignant B cells were treated using pH sensitive dye doped NPs and pH sensitive dye doped NP1 in order to assess interaction of nanoparticles with cells. Uptake measurements were performed through quantification of the fluorescence of the pH sensitive dye. As proof of concept, Fludarabine was then incorporated on to the surface of NPs in order to investigate its potential as a nanotherapeutic. Cytotoxicity studies were performed using flow cytometric analysis mentioned above following a 24 hour incubation. Results and Conclusions Quantitative and qualitative analysis identified uptake of NPs by normal and malignant B-lymphocytes with optimal NPs concentration for uptake determined at 25 μg/ml. Non-functionalised NPs in the range of 15-50nm were internalised by cells. There was a notable decrease in the interaction of NPs with cells under physiologically relevant fluid shear flow in comparison to static conditions, resulting in a corresponding decrease in uptake, highlighting the rationale for a CLL cell-targeted NP. The results of the adhesion experiment using I-83 cells and patient derived CLL cells to the HUVEC monolayer in a micro-fluidics system showed that patient CLL adhesion decreased after NP treatment (p=0.01, n=3). Cytotoxicity studies show that exposure to uncoated Fe2O3 nanoparticles yields an IC50 value of 23μg/mL +/- 5 μg/mL in comparison to coated, stabilized Fe2O3 nanoparticles with an IC50 of 49μg/mL +/- 5 μg/mL. Functionalisation of NPs with CD52 antibody (NP-1) resulted in significantly increased uptake (p<0.0001, n=3) and cytotoxicity. Preparation of these nanoparticles was reproducible and the particles remained stable in suspension for over 4 weeks. Cells treated with NPs bound Fludarabine were found to have significantly increased cytotoxicity in comparison to stabilized NPs (IC50 of 21μg/mL +/- 1μg/mL. In summary, this work provides proof of concept of efficacy for a targeted nanotherapeutic in haematological malignancies. Disclosures No relevant conflicts of interest to declare.

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Aquaporin-1: a major modulator of aging-mediated platelet/endothelial dysfunction in atherothrombosis

European Heart Journal ◽

10.1093/ehjci/ehaa946.3788 ◽

2020 ◽

Vol 41 (Supplement_2) ◽

Author(s):

S.S Saeedi Saravi ◽

G.G Camici ◽

T.F Luscher ◽

J.H Beer

Keyword(s):

Endothelial Cells ◽

Platelet Aggregation ◽

Endothelial Dysfunction ◽

Shear Flow ◽

Platelet Adhesion ◽

Funding Source ◽

Rolling Velocity ◽

Caveolin 1 ◽

Aquaporin 1 ◽

Aqp1 Expression

Abstract Background Aging is associated with development of cardiovascular diseases, including atherothrombosis. Aquaporin-1 (AQP1) is a water channel that also transports hydrogen peroxide (H2O2). Regarding to the role of oxidative stress in atherothrombosis, we hypothesized that AQP1 modulates aging-associated platelet/endothelial dysfunction. Methods Human aortic endothelial cells (HAEC) from passages 5 (young) to 15 (senescent/old) were subjected to fluorescent immunocytochemistry to detect AQP1 protein expression and subcellular localization. The cells were also probed for AQP1 and the phospho/total proteins (AMPK, acetyl-coA-carboxylase (ACC), caveolin-1 and eNOS) for studying the signal transduction by immunoblotting. The endothelial cells were transfected with constructs containing H2O2 biosensor HyPer targeted to cell nucleus or cytosol followed by fluorescence imaging. The transcriptional levels of pro-inflammatory/pro-atherogenic vs. anti-inflammatory/atheroprotective genes in the cells were assessed by qRT-PCR. Human blood samples were taken and treated with or without AQP1 inhibitor (Bacopaside II, 10 μM) to examine the platelet adhesion and rolling velocity on vWF under high shear flow (100 dyn/cm2). Also, platelet aggregation in response to collagen (2 μM), ADP (1 μM) and TRAP (1 μM) were recorded. Results First, the senescence of HAEC was adjusted by a significant increase in β-galactosidase activity from passage 5 to 15. AQP1 immunofluorescence showed a remarkable increase in the young (P.5) compared to senescent (P.15) cells. Immunoblot analyses showed that aging leads to significant increases in AQP1 intensity and phosphorylation of caveolin-1 (Tyr14) and ACC (Ser79), along with decreases in phosphorylation of eNOS (Ser1177) and AMPK (Thr172) (p<0.01, n>6). Fluorescence imaging documented a robust H2O2 production in the senescent endothelial cell cytosol, but not nucleus, and activated TNF-α gene, whereas the transcription of hemoxygenase-1 gene enhanced in the young cells (p<0.01). AQP1 inhibition reduced platelet adhesion and thrombus formation, and elevated platelet rolling velocity on vWF under shear flow (p<0.01). Also, a decrease was found in platelet aggregation in response to AQP1 inhibition (p<0.05). Conclusion These studies, for the first time, demonstrate that aging induces AQP1 expression in endothelial cells and platelets, and modulates the dephosphorylation of AMPK/eNOS. These may lead to platelet/endothelial dysfunction and production of pro-coagulant/pro-inflammatory factors via ACC activation. Therefore, AQP1 inhibition could potentially be exploited as a therapeutic strategy for improving age-related atherothrombosis. Figure 1. Aging increases AQP1 expression in HAEC that leads to dephosphorylation of AMPK and eNOS. AQP inhibition also improves platelet function. *p<0.05, **p<0.01 compared to control. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Swiss National Science Foundation (SNSF)

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