scholarly journals Resistance of endothelial cells to SARS-CoV-2 infection in vitro

2020 ◽  
Author(s):  
Blerina Ahmetaj-Shala ◽  
Thomas P. Peacock ◽  
Laury Baillon ◽  
Olivia C. Swann ◽  
Hime Gashaw ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Epithelial Cells ◽  
Cell Lines ◽  
Post Infection ◽  
Clinical Syndrome ◽  
The Body ◽  
Human Embryonic Kidney Cells ◽  
Nasal Epithelial Cells

AbstractRationaleThe secondary thrombotic/vascular clinical syndrome of COVID-19 suggests that SARS-CoV-2 infects not only respiratory epithelium but also the endothelium activating thrombotic pathways, disrupting barrier function and allowing access of the virus to other organs of the body. However, a direct test of susceptibility to SARS-CoV-2 of authentic endothelial cell lines has not been performed.ObjectiveTo determine infectibility of primary endothelial cell lines with live SARS-CoV-2 and pseudoviruses expressing SARS-CoV-2 spike protein.Methods and ResultsExpression of ACE2 and BSG pathways genes was determined in three types of endothelial cells; blood outgrowth, lung microvascular and aortic endothelial cells. For comparison nasal epithelial cells, Vero E6 cells (primate kidney fibroblast cell line) and HEK 293T cells (human embryonic kidney cells) transfected with either ACE2 or BSG were used as controls. Endothelial and Vero E6 cells were treated with live SARS-CoV-2 virus for 1 hour and imaged at 24 and 72 hours post infection. Pseudoviruses containing SARS-CoV-2, Ebola and Vesicular Stomatis Virus glycoproteins were generated and added to endothelial cells and HEK 239Ts for 2 hours and infection measured using luminescence at 48 hours post infection. Compared to nasal epithelial cells, endothelial cells expressed low or undetectable levels of ACE2 and TMPRSS2 but comparable levels of BSG, PPIA and PPIB. Endothelial cells showed no susceptibility to live SARS-CoV-2 or SARS-CoV-2 pseudovirus (but showed susceptibility to Ebola and Vesicular Stomatitis Virus). Overexpression of ACE2 but not BSG in HEK 239T cells conferred SARS-CoV-2 pseudovirus entry. Endothelial cells primed with IL-1ß remained resistant to SARS-CoV-2.ConclusionEndothelial cells are resistant to infection with SARS-CoV-2 virus, in line with relatively low levels of ACE2 and TMPRSS2, suggesting that the vascular dysfunction and thrombosis seen in severe COVID-19 is a result of factors released by adjacent infected cells (e.g. epithelial cells) and/or circulating, systemic inflammatory mediators.

scholarly journals Identification of urban particulate matter-induced disruption of human respiratory mucosa integrity using whole transcriptome analysis and organ-on-a chip

2019 ◽  
Vol 13 (1) ◽  
Author(s):  
Junhyoung Byun ◽  
Boa Song ◽  
Kyungwoo Lee ◽  
Byoungjae Kim ◽  
Hae Won Hwang ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Particulate Matter ◽  
Epithelial Cells ◽  
Respiratory Mucosa ◽  
Nasal Epithelial Cells ◽  
Tissue Microenvironment ◽  
Human Nasal Epithelial Cells ◽  
Whole Transcriptome Analysis ◽  
Whole Transcriptome

Abstract Background Exposure to air particulate matter (PM) is associated with various diseases in the human respiratory system. To date, most in vitro studies showing cellular responses to PM have been performed in cell culture using a single cell type. There are few studies considering how multicellular networks communicate in a tissue microenvironment when responding to the presence of PM. Here, an in vitro three-dimensional (3D) respiratory mucosa-on-a-chip, composed of human nasal epithelial cells, fibroblasts, and endothelial cells, is used to recapitulate and better understand the effects of urban particulate matter (UPM) on human respiratory mucosa. Results We hypothesized that the first cells to contact with UPM, the nasal epithelial cells, would respond similar to the tissue microenvironment, and the 3D respiratory mucosa model would be a suitable platform to capture these events. First, whole transcriptome analysis revealed that UPM induced gene expression alterations in inflammatory and adhesion-related genes in human nasal epithelial cells. Next, we developed an in vitro 3D respiratory mucosa model composed of human nasal epithelial cells, fibroblasts, and endothelial cells and demonstrated that the model is structurally and functionally compatible with the respiratory mucosa. Finally, we used our model to expose human nasal epithelial cells to UPM, which led to a disruption in the integrity of the respiratory mucosa by decreasing the expression of zonula occludens-1 in both the epithelium and endothelium, while also reducing vascular endothelial cadherin expression in the endothelium. Conclusions We demonstrate the potential of the 3D respiratory mucosa model as a valuable tool for the simultaneous evaluation of multicellular responses caused by external stimuli in the human respiratory mucosa. We believe that the evaluation strategy proposed in the study will move us toward a better understanding of the detailed molecular mechanisms associated with pathological changes in the human respiratory system.

scholarly journals Human Cytomegalovirus Cell Tropism and Host Cell Receptors

Vaccines ◽  
2019 ◽  
Vol 7 (3) ◽  
pp. 70 ◽  
Author(s):  
Gerna ◽  
Kabanova ◽  
Lilleri
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Epithelial Cells ◽  
Human Cytomegalovirus ◽  
Current Data ◽  
Immunosuppressed Patient ◽  
Cell Tropism ◽  
Virus Envelope

In the 1970s–1980s, a striking increase in the number of disseminated human cytomegalovirus (HCMV) infections occurred in immunosuppressed patient populations. Autopsy findings documented the in vivo disseminated infection (besides fibroblasts) of epithelial cells, endothelial cells, and polymorphonuclear leukocytes. As a result, multiple diagnostic assays, such as quantification of HCMV antigenemia (pp65), viremia (infectious virus), and DNAemia (HCMV DNA) in patient blood, were developed. In vitro experiments showed that only low passage or endothelial cell-passaged clinical isolates, and not laboratory-adapted strains, could reproduce both HCMV leuko- and endothelial cell-tropism, which were found through genetic analysis to require the three viral genes UL128, UL130, and UL131 of the HCMV UL128 locus (UL128L). Products of this locus, together with gH/gL, were shown to form the gH/gL/pUL128L pentamer complex (PC) required for infection of epithelial cells/endothelial cells, whereas gH/gL and gO form the gH/gL/gO trimer complex (TC) required for infection of all cell types. In 2016, following previous work, a receptor for the TC that mediates entry into fibroblasts was identified as PDGFRα, while in 2018, a receptor for the PC that mediates entry into endothelial/epithelial cells was identified as neuropilin2 (Nrp2). Furthermore, the olfactory receptor family member OR14I1 was recently identified as a possible additional receptor for the PC in epithelial cells. Thus, current data support two models of viral entry: (i) in fibroblasts, following interaction of PDGFRα with TC, the latter activates gB to fuse the virus envelope with the cell membrane, whereas (ii) in epithelial cells/endothelial cells, interaction of Nrp2 (and OR14I1) with PC promotes endocytosis of virus particles, followed by gB activation by gH/gL/gO (or gH/gL) and final low-pH entry into the cell.

scholarly journals Perturbations in the fibrinolytic pathway abolish cyst formation but not capillary-like organization of cultured murine endothelial cells

Blood ◽  
1994 ◽  
Vol 83 (11) ◽  
pp. 3206-3217 ◽  
Author(s):  
N Dubois-Stringfellow ◽  
A Jonczyk ◽  
VL Bautch
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Basement Membrane ◽  
Organizational Behavior ◽  
Cell Lines ◽  
Primary Cultures ◽  
Cyst Formation ◽  
Fibrin Gels

Abstract Fibrinolytic activity and its relation to morphogenesis was investigated in several transformed murine endothelial cell lines and primary cultures of endothelial cells. Two in vitro systems, fibrin gels and Matrigel (Collaborative Research, Bedford, MA), were used. Fibrin gels model a fibrin-rich extracellular matrix that frequently supports neovascularization in vivo, and Matrigel models the basement membrane surrounding quiescent endothelial cells in vivo. The transformed endothelial cell lines have higher levels of plasminogen activator (PA) mRNA than primary cultures of endothelial cells, and an increased PA-mediated proteolytic activity was correlated with formation of cysts in fibrin gels. Addition of neutralizing anti- urokinase antibodies, plasminogen depletion, or addition of a plasmin inhibitor prevented cyst formation. Addition of plasminogen restored the ability to form cysts in the plasminogen-depleted system. Normal endothelial cells organized into capillary-like structures in fibrin gels regardless of manipulations affecting the fibrinolytic pathway. In Matrigel, both transformed and primary cultures of endothelial cells rapidly formed a capillary-like network that was not affected by plasminogen depletion or addition of plasmin inhibitors. Thus, elements of the fibrinolytic pathway necessary for cyst formation are not critical in capillary-like structure formation on a reconstituted basement membrane. These results suggest that plasmin is essential for hemangioma formation but is not critical to the organizational behavior of normal endothelial cells.

scholarly journals Angiogenic microenvironment augments impaired endothelial responses under diabetic conditions

2014 ◽  
Vol 306 (8) ◽  
pp. C768-C778 ◽  
Author(s):  
Abdul Q. Sheikh ◽  
Courtney Kuesel ◽  
Toloo Taghian ◽  
Jennifer R. Hurley ◽  
Wei Huang ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
High Glucose ◽  
Microvascular Complications ◽  
The Body ◽  
Type I ◽  
Chronic Hyperglycemia ◽  
Biomechanical Responses ◽  
Acute And Chronic Exposure

Diabetes-induced cardiomyopathy is characterized by cardiac remodeling, fibrosis, and endothelial dysfunction, with no treatment options currently available. Hyperglycemic memory by endothelial cells may play the key role in microvascular complications in diabetes, providing a potential target for therapeutic approaches. This study tested the hypothesis that a proangiogenic environment can augment diabetes-induced deficiencies in endothelial cell angiogenic and biomechanical responses. Endothelial responses were quantified for two models of diabetic conditions: 1) an in vitro acute and chronic hyperglycemia where normal cardiac endothelial cells were exposed to high-glucose media, and 2) an in vivo chronic diabetes model where the cells were isolated from rats with type I streptozotocin-induced diabetes. Capillary morphogenesis, VEGF and nitric oxide expression, cell morphology, orientation, proliferation, and apoptosis were determined for cells cultured on Matrigel or proangiogenic nanofiber hydrogel. The effects of biomechanical stimulation were assessed following cell exposure to uniaxial strain. The results demonstrate that diabetes alters cardiac endothelium angiogenic response, with differential effects of acute and chronic exposure to high-glucose conditions, consistent with the concept that endothelial cells may have a long-term “hyperglycemic memory” of the physiological environment in the body. Furthermore, endothelial cell exposure to strain significantly diminishes their angiogenic potential following strain application. Both diabetes and strain-associated deficiencies can be augmented in the proangiogenic nanofiber microenvironment. These findings may contribute to the development of novel approaches to reverse hyperglycemic memory of endothelium and enhance vascularization of the diabetic heart, where improved angiogenic and biomechanical responses can be the key factor to successful therapy.

scholarly journals Bovine Organospecific Microvascular Endothelial Cell Lines as New and Relevant In Vitro Models to Study Viral Infections

2020 ◽  
Vol 21 (15) ◽  
pp. 5249 ◽  
Author(s):  
Anne-Claire Lagrée ◽  
Fabienne Fasani ◽  
Clotilde Rouxel ◽  
Marine Pivet ◽  
Marie Pourcelot ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Cell Lines ◽  
T Antigen ◽  
In Vitro Studies ◽  
Microvascular Endothelial Cell ◽  
Target Cells ◽  
Microvascular Endothelial

Microvascular endothelial cells constitute potential targets for exogenous microorganisms, in particular for vector-borne pathogens. Their phenotypic and functional variations according to the organs they are coming from provide an explanation of the organ selectivity expressed in vivo by pathogens. In order to make available relevant tools for in vitro studies of infection mechanisms, our aim was to immortalize bovine organospecific endothelial cells but also to assess their permissivity to viral infection. Using transfection with SV40 large T antigen, six bovine microvascular endothelial cell lines from various organs and one macrovascular cell line from an umbilical cord were established. They display their own panel of endothelial progenitor/mature markers, as assessed by flow cytometry and RT-qPCR, as well as the typical angiogenesis capacity. Using both Bluetongue and foot-and-mouth disease viruses, we demonstrate that some cell lines are preferentially infected. In addition, they can be transfected and are able to express viral proteins such as BTV8-NS3. Such microvascular endothelial cell lines bring innovative tools for in vitro studies of infection by viruses or bacteria, allowing for the study of host-pathogen interaction mechanisms with the actual in vivo target cells. They are also suitable for applications linked to microvascularization, such as anti-angiogenic and anti-tumor research, growing fields in veterinary medicine.

scholarly journals Perturbations in the fibrinolytic pathway abolish cyst formation but not capillary-like organization of cultured murine endothelial cells

Blood ◽  
1994 ◽  
Vol 83 (11) ◽  
pp. 3206-3217 ◽  
Author(s):  
N Dubois-Stringfellow ◽  
A Jonczyk ◽  
VL Bautch
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Basement Membrane ◽  
Organizational Behavior ◽  
Cell Lines ◽  
Primary Cultures ◽  
Cyst Formation ◽  
Fibrin Gels

Fibrinolytic activity and its relation to morphogenesis was investigated in several transformed murine endothelial cell lines and primary cultures of endothelial cells. Two in vitro systems, fibrin gels and Matrigel (Collaborative Research, Bedford, MA), were used. Fibrin gels model a fibrin-rich extracellular matrix that frequently supports neovascularization in vivo, and Matrigel models the basement membrane surrounding quiescent endothelial cells in vivo. The transformed endothelial cell lines have higher levels of plasminogen activator (PA) mRNA than primary cultures of endothelial cells, and an increased PA-mediated proteolytic activity was correlated with formation of cysts in fibrin gels. Addition of neutralizing anti- urokinase antibodies, plasminogen depletion, or addition of a plasmin inhibitor prevented cyst formation. Addition of plasminogen restored the ability to form cysts in the plasminogen-depleted system. Normal endothelial cells organized into capillary-like structures in fibrin gels regardless of manipulations affecting the fibrinolytic pathway. In Matrigel, both transformed and primary cultures of endothelial cells rapidly formed a capillary-like network that was not affected by plasminogen depletion or addition of plasmin inhibitors. Thus, elements of the fibrinolytic pathway necessary for cyst formation are not critical in capillary-like structure formation on a reconstituted basement membrane. These results suggest that plasmin is essential for hemangioma formation but is not critical to the organizational behavior of normal endothelial cells.

scholarly journals SARS-CoV-2 spike protein induces paracrine senescence and leukocyte adhesionin endothelial cells

2021 ◽  
Author(s):  
Keith Meyer ◽  
Tapas Patra ◽  
Vijayamahantesh ◽  
Ranjit Ray
Keyword(s):  
Cell Culture ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Epithelial Cells ◽  
Protein Expression ◽  
Cell Lines ◽  
Cellular Senescence ◽  
Leukocyte Adhesion ◽  
Spike Protein ◽  
Cell Culture Supernatant

Increased mortality in COVID-19 often associates with microvascular complications. We have recently shown that SARS-CoV-2 spike protein promotes an inflammatory cytokine IL-6/IL-6R induced trans-signaling response and alarmin secretion. Virus infected or spike transfected human epithelial cells exhibited an increase in senescence state with the release of senescence associated secretory proteins (SASP) related inflammatory molecules. Introduction of BRD4 inhibitor AZD5153 to senescent epithelial cells reversed this effect and reduced SASP related inflammatory molecule release in TMNK-1 or EA hy926 as representative human endothelial cell line, when exposed to cell culture medium (CM) derived from A549 cells expressing SARS-CoV-2 spike protein, also exhibited a senescence phenotype with enhanced p16, p21, SA-β-galactosidase expression, and triggered SASP pathways. Inhibition of IL-6 trans-signaling by Tocilizumab and inhibition of inflammatory receptor signaling by the BTK inhibitor Zanubrutinib, prior to exposure of CM to endothelial cells, inhibited p21 and p16 induction. We also observed an increase in reactive oxygen species (ROS) in A549 spike transfected and endothelial cells exposed to spike transfected CM. ROS generation in endothelial cell lines was reduced after treatment with Tocilizumab and Zanubrutinib. Cellular senescence was associated with an increased level of the endothelial adhesion molecules, VCAM-1 and ICAM-1 with in vitro leukocyte attachment potential. Inhibition of senescence or SASP function prevented VCAM-1/ICAM-1 expression and leukocyte attachment. Taken together, we identified that the exposure of human endothelial cells to cell culture supernatant derived from SARS-CoV-2 spike protein expression displayed cellular senescence markers, leading to enhanced leukocyte adhesion. Importance: The present study was aimed at examining the underlying mechanism of extrapulmonary manifestations of SARS-CoV-2 spike protein associated pathogenesis, with the notion that infection of the pulmonary epithelium can lead to mediators that drive endothelial dysfunction. We utilized SARS-CoV-2 spike protein expression in cultured cells of human hepatocytes (Huh7.5) and pneumocytes (A549) to generate conditioned culture media (CM). Endothelial cell lines (TMNK-1 or EA hy926 ) treated with CM exhibited increase in cellular senescence markers by a paracrine mode, and lead to leukocyte adhesion. Overall, the link between these responses in endothelial cell senescence, and a potential contribution to microvascular complication in productively SARS-CoV-2 infected humans is implicated. Furthermore, the use of inhibitors (BTK, IL-6 and BRD4) showed reverse effect in the senescent cells. These results may support the selection of potential adjunct therapeutic modalities to impede SARS-CoV-2 associated pathogenesis.

Endothelial Cell Adhesion on Highly Controllable Compared to Random Nanostructured Titanium Surface Features

2006 ◽  
Vol 951 ◽  
Author(s):  
Jing Lu ◽  
Thomas J. Webster
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
In Vitro Study ◽  
The Body ◽  
Vitro Study ◽  
Nanostructured Surface ◽  
Vascular Stent ◽  
Surface Features ◽  
Vascular Stents

ABSTRACTThe application of vascular stents using conventional metals is limited because the implantation process will cause significant injury to the vascular wall and endothelium, resulting in neointima hyperplasia and then the development of long-term restenosis. The objective of this in vitro study was to investigate endothelial cell function (especially their adhesion behavior) on highly controllable nanostructured surface features. Considering the importance of the endothelium and its properties, highly controllable nanostructured surface features of titanium (a popular vascular stent metal) were created using E-beam evaporation to promote endothelialization and to control the direction of endothelial cells on vascular stents. Endothelial cells are aligned with blood flow naturally in the body. In this manner, the present in vitro study provides much promise for the use of nanotechnology for improving metals for vascular stent applications.

scholarly journals Neutrophil-Derived Microvesicle Induced Dysfunction of Brain Microvascular Endothelial Cells In Vitro

2019 ◽  
Vol 20 (20) ◽  
pp. 5227 ◽  
Author(s):  
Anjana Ajikumar ◽  
Merete B. Long ◽  
Paul R. Heath ◽  
Stephen B. Wharton ◽  
Paul G. Ince ◽  
...  
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
The Body ◽  
Confocal Imaging ◽  
Microvascular Endothelial Cells ◽  
Microvascular Endothelial Cell ◽  
Brain Microvascular Endothelial Cells ◽  
Microvascular Endothelial

The blood-brain barrier (BBB), composed of brain microvascular endothelial cells (BMEC) that are tightly linked by tight junction (TJ) proteins, restricts the movement of molecules between the periphery and the central nervous system. Elevated systemic levels of neutrophils have been detected in patients with altered BBB function, but the role of neutrophils in BMEC dysfunction is unknown. Neutrophils are key players of the immune response and, when activated, produce neutrophil-derived microvesicles (NMV). NMV have been shown to impact the integrity of endothelial cells throughout the body and we hypothesize that NMV released from circulating neutrophils interact with BMEC and induce endothelial cell dysfunction. Therefore, the current study investigated the interaction of NMV with human BMEC and determined whether they altered gene expression and function in vitro. Using flow cytometry and confocal imaging, NMV were shown to be internalized by the human cerebral microvascular endothelial cell line hCMEC/D3 via a variety of energy-dependent mechanisms, including endocytosis and macropinocytosis. The internalization of NMV significantly altered the transcriptomic profile of hCMEC/D3, specifically inducing the dysregulation of genes associated with TJ, ubiquitin-mediated proteolysis and vesicular transport. Functional studies confirmed NMV significantly increased permeability and decreased the transendothelial electrical resistance (TEER) of a confluent monolayer of hCMEC/D3. These findings indicate that NMV interact with and affect gene expression of BMEC as well as impacting their integrity. We conclude that NMV may play an important role in modulating the permeability of BBB during an infection.

scholarly journals Immune response and pathogen invasion at the choroid plexus in the onset of cerebral toxoplasmosis

2022 ◽  
Vol 19 (1) ◽  
Author(s):  
Caio Andreeta Figueiredo ◽  
Johannes Steffen ◽  
Lorena Morton ◽  
Sushmitha Arumugam ◽  
Oliver Liesenfeld ◽  
...  
Keyword(s):  
Immune Response ◽  
Endothelial Cells ◽  
Epithelial Cells ◽  
Choroid Plexus ◽  
The Body ◽  
Potential Contribution ◽  
Pathogen Invasion ◽  
The Impact

Abstract Background Toxoplasma gondii (T. gondii) is a highly successful parasite being able to cross all biological barriers of the body, finally reaching the central nervous system (CNS). Previous studies have highlighted the critical involvement of the blood–brain barrier (BBB) during T. gondii invasion and development of subsequent neuroinflammation. Still, the potential contribution of the choroid plexus (CP), the main structure forming the blood–cerebrospinal fluid (CSF) barrier (BCSFB) have not been addressed. Methods To investigate T. gondii invasion at the onset of neuroinflammation, the CP and brain microvessels (BMV) were isolated and analyzed for parasite burden. Additionally, immuno-stained brain sections and three-dimensional whole mount preparations were evaluated for parasite localization and morphological alterations. Activation of choroidal and brain endothelial cells were characterized by flow cytometry. To evaluate the impact of early immune responses on CP and BMV, expression levels of inflammatory mediators, tight junctions (TJ) and matrix metalloproteinases (MMPs) were quantified. Additionally, FITC-dextran was applied to determine infection-related changes in BCSFB permeability. Finally, the response of primary CP epithelial cells to T. gondii parasites was tested in vitro. Results Here we revealed that endothelial cells in the CP are initially infected by T. gondii, and become activated prior to BBB endothelial cells indicated by MHCII upregulation. Additionally, CP elicited early local immune response with upregulation of IFN-γ, TNF, IL-6, host-defence factors as well as swift expression of CXCL9 chemokine, when compared to the BMV. Consequently, we uncovered distinct TJ disturbances of claudins, associated with upregulation of MMP-8 and MMP-13 expression in infected CP in vivo, which was confirmed by in vitro infection of primary CP epithelial cells. Notably, we detected early barrier damage and functional loss by increased BCSFB permeability to FITC-dextran in vivo, which was extended over the infection course. Conclusions Altogether, our data reveal a close interaction between T. gondii infection at the CP and the impairment of the BCSFB function indicating that infection-related neuroinflammation is initiated in the CP.

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