scholarly journals SARS-CoV-2 spike protein expressing epithelial cells promotes senescence associated secretory phenotype in endothelial cells and increased inflammatory response

2021 ◽  
Author(s):  
Keith Meyer ◽  
Tapas Patra ◽  
Vijay Mahantesh ◽  
Ranjit Ray
Keyword(s):  
Cell Culture ◽  
Endothelial Cells ◽  
Epithelial Cells ◽  
Culture Supernatant ◽  
Microvascular Complications ◽  
Specific Inhibitor ◽  
Spike Protein ◽  
Cell Culture Supernatant ◽  
Human Endothelial Cells ◽  
Secretory Phenotype

AbstractIncreased mortality in COVID-19 often associates with thrombotic and microvascular complications. We have recently shown that SARS-CoV-2 spike protein promotes inflammatory cytokine IL-6/IL-6R induced trans-signaling responses which modulate MCP-1 expression in human endothelial cells. MCP-1 is secreted as a major component of the senescence associated secretory phenotype (SASP). Virus infected or Spike transfected human pulmonary epithelial cells (A549) exhibited an increase in senescence related marker proteins. TMNK; as a representative human endothelial cell line, when exposed to cell culture supernatant derived from A549 cells expressing SARS-CoV-2 spike protein (Spike CM) exhibited a senescence phenotype with enhanced p16, p21, and SA-β-galactosidase expression. Inhibition of IL-6 trans-signaling by Tocilizumab, prior to exposure of supernatant to endothelial cells, inhibited p16 and p21 induction. Likewise, inhibition of receptor signaling by Zanabrutinib or Brd4 function by AZD5153 also led to limited induction of p16 expression. Senescence lead to an enhanced level of adhesion molecule, ICAM-1 and VCAM-1 in human endothelial cells, and TPH1 attachment by in vitro assay. Inhibition of senescence or SASP function prevented ICAM/VCAM expression and leukocyte attachment. We also observed an increase in oxidative stress in A549 spike transfected and endothelial cells exposed to Spike CM. ROS generation in TMNK was reduced after treatment with the IL-6 specific inhibitor Tociliximab, and with the specific inhibitors Zanabrutinib and AZD5153. 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 with coronary blockade potential.

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.

scholarly journals Endothelin Receptor Down-Regulation Mediated Ligand Regulation Mechanisms Protect Against Cellular Hypoxia Injury in Rat Vascular Endothelial Cells

2016 ◽  
Vol 40 (6) ◽  
pp. 1443-1454 ◽  
Author(s):  
Long Li ◽  
Mushuang Hu ◽  
Long Zheng ◽  
Chao Zhang ◽  
Jiawei Li ◽  
...  
Keyword(s):  
Cell Culture ◽  
Endothelial Cells ◽  
Culture Supernatant ◽  
Cellular Protein ◽  
Oxygen Deprivation ◽  
Cell Culture Supernatant ◽  
Vascular Endothelial ◽  
Expression Levels ◽  
Cellular Hypoxia ◽  
Sirna Group

Objective: Investigation of the effect of endothelin receptor A (ETaR)-targeting small interfering RNA (siRNA) on rat vascular endothelial cellular hypoxia injury, as well as its underlying mechanism. Methods: An in vitro rat vascular smooth muscle cells - endothelial cells co-culture model was established and transfected with ETaR siRNA before hypoxia treatment. Cell culture supernatant, cellular protein and RNA were collected and examined at 0.5hrs, 1hrs, 2hrs, 4hrs, 8hrs, 16hrs, 24hrs and 48hrs of hypoxia with 1% oxygen. The time point at which the best silencing effect was achieved was chosen, eNOS inhibitor L-NAME was added, and post hypoxia cell culture supernatant, cellular protein and RNA was collected for further examination. Results: After hypoxic treatment, endothelial-1 (ET-1) and ETaR expression levels gradually increased as oxygen deprivation extended. ET-1 and ETaR expression levels were significantly lower in the ETaR siRNA group compared with the Hypoxia group (P<0.001). Such difference peaked at 4hrs of hypoxia. ELISA examination of cell culture supernatant revealed that the amount of ET-1 and TGF-βin the ETaR siRNA group were significantly lower compared to the Hypoxia group at all times, while the amount of NO and eNOS was higher. After 4 hrs of hypoxia, Smad2, Smad3, HIF-1, TNF-α, IFN-γ, IL-6, MCP-1, NF-κb, ET-1 and ANG II mRNA expression in endothelial cells and ETaR mRNA expression in A-10 cells of the ETaR siRNA group were lower than those of the Hypoxia siRNA group, while such results were much higher in the L-NAME group. Western Blot results showed lower expression of ETaR in the ETaR siRNA group compared with the hypoxia and negative siRNA groups, as well as significantly higher ETaR expression in the L-NAME group compared with the ETaR siRNA group. PI3K and p-AKT expression levels were mildly elevated after mild oxygen deprivation, and ETaR siRNA was able to enhance such elevation induced by hypoxia. In the L-NAME group, PI3K and p-AKT expression was much higher than the ETaR siRNA group. PKG and sGC expression levels significantly descended after mild oxygen deprivation. While such levels were higher in the ETaR siRNA group, compared with the hypoxia and negative siRNA groups, the L-NAME group had lower levels of PKG and sGC compared with the ETaR siRNA group. Conclusion: ETaR siRNA is capable of down-regulating the expression of inflammatory and transcription factors among endothelial cells treated with hypoxia. Down-regulation of ET-1 is triggered by altered nucleus transcription factor activity through the sGC/PKG signal pathway, and results in enhanced eNOS activity through the PI3K/Akt signal pathway. We suspect this to be the mechanism of the protective effect of ETaR siRNA.

Specificity of the Thrombin-Induced Release of Tissue Plasminogen Activator from Cultured Human Endothelial Cells

1986 ◽  
Vol 56 (02) ◽  
pp. 115-119 ◽  
Author(s):  
Eugene G Levin ◽  
David M Stern ◽  
Peter P Nawroth ◽  
Richard A Marlar ◽  
Daryl S Fair ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Protein C ◽  
Primary Cultures ◽  
Activated Protein C ◽  
Specific Inhibitor ◽  
Factor Xa ◽  
Human Endothelial Cells ◽  
Tissue Plasminogen

SummaryThe addition of thrombin (9 nM) to primary cultures of human endothelial cells induces a 6- to 7-fold increase in the rate of release of tissue plasminogen activator (tPA). Several other serine proteases which specifically interact with endothelial cells were also analyzed for their effect on tPA release. Gamma-thrombin, an autocatalytic product of α-thrombin, promoted tPA release but was less effective than α-thrombin. A maximum increase of 5.5-fold was observed, although a concentration of γ-thrombin 20 times greater than α-thrombin was required. The response to Factor Xa was similar to α-thrombin, although the stimulation was significantly reduced by the addition of hirudin or DAPA suggesting that prothrombin activation was occurring. The simultaneous addition of prothrombin with Factor Xa resulted in enhanced tPA release equal to that observed with an equimolar concentration of active α-thrombin. Thus, under these conditions, Factor Xa-cell surface mediated activation of prothrombin can lead to a secondary effect resulting from cell-thrombin interaction. Activated protein C, which has been implicated as a profibrinolytic agent, was also tested. No change in tPA release occurred after the addition of up to 325 nM activated protein C in the presence or absence of proteins. Factor IXa and plasmin were also ineffective. The effect of thrombin on the endothelial cell derived plasminogen activator specific inhibitor was also studied. Thrombin produced a small but variable release of the inhibitor with an increase of less than twice that of non-thrombin treated controls.

Continuous purification of antibodies from cell culture supernatant with aqueous two-phase systems: From concept to process

2013 ◽  
Vol 8 (3) ◽  
pp. 352-362 ◽  
Author(s):  
Paula A. J. Rosa ◽  
Ana M. Azevedo ◽  
S. Sommerfeld ◽  
Martina Mutter ◽  
Werner Bäcker ◽  
...  
Keyword(s):  
Cell Culture ◽  
Culture Supernatant ◽  
Cell Culture Supernatant ◽  
Two Phase ◽  
Aqueous Two Phase Systems ◽  
Phase Systems

Abstract MP30: Circulating Sars-cov-2 Spike Protein 1 Causes Microarteriolar Oxidative Stress, Endothelial Dysfunction And Enhanced Thromboxane And Endothelin Contractility That Are Prevented By Spironolactone.

Hypertension ◽  
2021 ◽  
Vol 78 (Suppl_1) ◽  
Author(s):  
Dan Wang ◽  
Christopher S Wilcox
Keyword(s):  
Oxidative Stress ◽  
Endothelial Cells ◽  
Endothelial Dysfunction ◽  
Fluorescence Microscopy ◽  
Vascular Dysfunction ◽  
Microvascular Complications ◽  
Spike Protein ◽  
Ros Generation ◽  
Intravenous Injections ◽  
Novel Model

Introduction and hypothesis: Following bodily entry, the SARS-CoV-2 virus undergoes pulmonary replication with release of circulating viral spike protein 1 (SP1) into the bloodstream. Uptake of SP1 by endothelial cells might provoke vascular dysfunction and thrombosis. We hypothesized that spironolactone could prevent microvascular complications from circulating SP1 in COVID-19. Methods: male C57Bl/6 mice received spironolactone (100 mg · kg -1 · d -1 PO x 3d) or vehicle and intravenous injections of recombinant full-length human SP1 (10 μg per mouse) or vehicle. They were euthanized after 3 days. Mesenteric resistant arterioles (n=4 per group) were dissected and mounted on isometric myographs. Acetylcholine-induced EDRF responses and L-NAME-inhibitable NO generation (DAF-FM fluorescence) were studied in pre-constricted vessels and contraction to endothelin 1 (ET1) or thromboxane (U-46, 619) and ET1-induced ROS (PEG-SOD inhibitable ethidium: dihydroethidium fluorescence) were studied by fluorescence microscopy in other vessels. Results: SP1 reduced acetylcholine-induced EDRF (17 ± 3 vs 27 ± 5 % mean ± sem; P < 0.05) and NO generation (0.21 ± 0.03 vs 0.36 ± 0.04, F 1 /F 0 ; P < 0.05) while increasing contraction to ET1 (10 -7 mol·l -1 : 124 ± 13 vs 89 ± 4 %; P < 0.05) and U-46, 619 (10 -6 mol·l -1 :114± 5 vs 87± 6 %; P < 0.05) and ET1-induced ROS generation(0.30± 0.08 vs 0.09± 0.03; P < 0.05). Spironolactone did not modify any of these responses in vessels from normal mice but prevented all the effects of SP1. Conclusion: these preliminary studies provide a novel model to study COVID-19 vasculopathy. They indicate that spironolactone can provide protection from microvascular oxidative stress, endothelial dysfunction and enhanced contractility and might thereby moderate COVID-19 complications.

scholarly journals Quantitation and properties of the active and latent plasminogen activator inhibitors in cultures of human endothelial cells

Blood ◽  
1986 ◽  
Vol 67 (5) ◽  
pp. 1309-1313 ◽  
Author(s):  
EG Levin
Keyword(s):  
Endothelial Cells ◽  
Conditioned Medium ◽  
Plasminogen Activator ◽  
Active Form ◽  
Specific Inhibitor ◽  
Human Endothelial Cells ◽  
Inhibitor Activity ◽  
Active Inhibitor ◽  
Latent Form ◽  
Cell Extracts

Abstract Human endothelial cells release two forms of a plasminogen activator- specific inhibitor: an active form that readily binds to and inhibits plasminogen activators and an inactive or latent form that has no anti- activator activity but which can be activated by denaturation. Latent and active forms of plasminogen activator-specific inhibitor were measured in cultures of human umbilical vein endothelial cells. Latent inhibitor was activated by treatment with 1% sodium dodecyl sulfate (SDS), and both forms were assayed by the 125I-fibrin plate method. After 16 hours, the conditioned medium contained 104.6 U/mL latent inhibitor activity and 2.6 U/mL active inhibitor. The level of each form in the culture medium increased with time although the activity associated with the latent form rose more rapidly: the ratio of latent to active inhibitor activity was 12 at four hours (10.3 U/mL v 0.86 U/mL) and reached 56 at 24 hours (155.3 U/mL v 2.80 U/mL). Intracellular inhibitor activity was associated with the active form only; no additional inhibitor activity was observed following SDS treatment of cell extracts. A decline in active inhibitor activity occurred during incubation at 37 degrees C with a 50% reduction in activity occurring in two hours. Treatment of conditioned medium with 10 U/mL thrombin also resulted in a loss of active inhibitor activity. The latent inhibitor, however, was not affected by either of these conditions. The inhibitor activity lost during incubation at 37 degrees C or thrombin treatment could be regenerated by SDS treatment, suggesting that the loss of the active inhibitor activity represented a conversion of this form to its latent counterpart. Thus, the concentration, stability, and regulation of these two forms of plasminogen activator inhibitor in human endothelial cell cultures differ significantly.

scholarly journals Insufficient Acidification of Autophagosomes Facilitates Group A Streptococcus Survival and Growth in Endothelial Cells

mBio ◽  
2015 ◽  
Vol 6 (5) ◽  
Author(s):  
Shiou-Ling Lu ◽  
Chih-Feng Kuo ◽  
Hao-Wen Chen ◽  
Yi-Shuan Yang ◽  
Ching-Chuan Liu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
Epithelial Cells ◽  
Bacterial Growth ◽  
Group A Streptococcus ◽  
Multiorgan Failure ◽  
Specific Inhibitor ◽  
Low Ph ◽  
Bafilomycin A1 ◽  
Group A

ABSTRACTGroup A streptococcus (GAS) is an important human pathogen, and its invasion via blood vessels is critically important in serious events such as bacteremia or multiorgan failure. Although GAS was identified as an extracellular bacterium, the internalization of GAS into nonphagocytic cells may provide a strategy to escape from immune surveillance and antibiotic killing. However, GAS has also been reported to induce autophagy and is efficiently killed within lysosome-fused autophagosomes in epithelial cells. In this study, we show that GAS can replicate in endothelial cells and that streptolysin O is required for GAS growth. Bacterial replication can be suppressed by altering GAS gene expression in an acidic medium before internalization into endothelial cells. The inhibitory effect on GAS replication can be reversed by treatment with bafilomycin A1, a specific inhibitor of vacuolar-type H+-ATPase. Compared with epithelial cells in which acidification causes autophagy-mediated clearance of GAS, there was a defect in acidification of GAS-containing vesicles in endothelial cells. Consequently, endothelial cells fail to maintain low pH in GAS-containing autophagosomes, thereby permitting GAS replication inside LAMP-1- and LC3-positive vesicles. Furthermore, treatment of epithelial cells with bafilomycin A1 resulted in defective GAS clearance by autophagy, with subsequent bacterial growth intracellularly. Therefore, low pH is a key factor for autophagy-mediated suppression of GAS growth inside epithelial cells, while defective acidification of GAS-containing vesicles results in bacterial growth in endothelial cells.IMPORTANCEPrevious reports showed that GAS can induce autophagy and is efficiently killed within lysosome-fused autophagosomes in epithelial cells. In endothelial cells, in contrast, induction of autophagy is not sufficient for GAS killing. In this study, we provide the first evidence that low pH is required to prevent intracellular growth of GAS in epithelial cells and that this mechanism is defective in endothelial cells. Treatment of GAS with low pH altered GAS growth rate and gene expression of virulence factors and resulted in enhanced susceptibility of GAS to intracellular lysosomal killing. Our findings reveal the existence of different mechanisms of host defense against GAS invasion between epithelial and endothelial cells.

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