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 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 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.

MO710GALECTIN-3, IS IT A NEW PLAYER IN PERITONEAL INFLAMMATION AMONG PATIENTS UNDERGOING PERITONEAL DIALYSIS?

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
Yael Einbinder ◽  
Keren Cohen-Hagai ◽  
Sydney Benchetrit ◽  
Tali Zitman-Gal
Keyword(s):  
Cell Culture ◽  
Peritoneal Dialysis ◽  
Endothelial Cell ◽  
Protein Expression ◽  
Inflammatory Process ◽  
Peritoneal Membrane ◽  
Peritoneal Equilibration Test ◽  
Endothelial Cell Culture ◽  
Dialysis Vintage ◽  
Mrna And Protein Expression

Abstract Background and Aims Peritoneal dialysis (PD) is a common used method for renal replacement therapy. Prolonged PD treatment causes structural and functional changes in the peritoneal membrane which are attributed to local inflammatory process in the peritoneal cavity. Galectin-3 (Gal-3) is a galactoside-binding lectin with pro-inflammatory and pro-fibrotic effects. The aim of this study was to assess correlation between Gal-3 serum and dialysate effluent levels with peritoneal membrane transport characteristics. Method Gal-3 levels in serum and dialysate effluent were measured simultaneously in prevalent PD patients in morning visit or during peritoneal equilibration test (PET). Gal-3 levels were correlated with clinical and laboratory parameters. Interlukin (IL) -6 levels were measured in dialysate effluent. Gal-3 mRNA and protein expression were evaluated after exposure of primary endothelial cell culture to several dialysate solutions. Results 37 PD patients were included in the study; mean age was 65.7±13.1 years, mean dialysis vintage was 17.5±13 months. Gal-3 levels in dialysate effluent correlated with peritoneal equilibration test (PET) results (0.663, p=0.005) and effluent IL-6 levels (0.674, p=0.002) but not with serum Gal-3 levels or dialysis vintage. Patients with high PET results had higher effluent Gal-3 levels as compared average low PET results. In multivariate regression analysis effluent IL-6 level was the most dominant predictor of effluent Gal-3 levels. Gal-3 mRNA and protein expression in primary endothelial cell culture were not affected by stimulation with dialysate solutions. Conclusion Our study demonstrated presence of Gal-3 within the dialysate effluent in PD patients. Gal-3 levels correlated with peritoneal membrane transport characteristics and effluent IL-6 levels suggesting a role in the inflammatory process within the peritoneal cavity.

The role of matrix metalloproteinase activity in the maturation of human capillary endothelial cells in vitro

1999 ◽  
Vol 112 (10) ◽  
pp. 1599-1609 ◽  
Author(s):  
B.M. Kraling ◽  
D.G. Wiederschain ◽  
T. Boehm ◽  
M. Rehn ◽  
J.B. Mulliken ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cell Proliferation ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Endothelial Cell Proliferation ◽  
Protein Levels ◽  
Matrix Deposition ◽  
Capillary Endothelial Cells ◽  
Vessel Maturation

Vessel maturation during angiogenesis (the formation of new blood vessels) is characterized by the deposition of new basement membrane and the downregulation of endothelial cell proliferation in the new vessels. Matrix remodeling plays a crucial, but still poorly understood role, in angiogenesis regulation. We present here a novel assay system with which to study the maturation of human capillary endothelial cells in vitro. When human dermal microvascular endothelial cells (HDMEC) were cultured in the presence of dibutyryl cAMP (Bt2) and hydrocortisone (HC), the deposition of a fibrous lattice of matrix molecules consisting of collagens type IV, type XVIII, laminin and thrombospondin was induced. In basal medium (without Bt2 and HC), HDMEC released active matrix metalloproteinases (MMPs) into the culture medium. However, MMP protein levels were significantly reduced by treatment with Bt2 and HC, while protein levels and activity of endogenous tissue inhibitor of MMPs (TIMP) increased. This shift in the proteolytic balance and matrix deposition was inhibited by the specific protein kinase A inhibitors RpcAMP and KT5720 or by substituting analogues without reported glucocorticoid activity for HC. The addition of MMP inhibitors human recombinant TIMP-1 or 1,10-phenanthroline to cultures under basal conditions induced matrix deposition in a dose-dependent manner, which was not observed with the serine protease inhibitor epsilon-amino-n-caproic acid (ACA). The deposited basement membrane-type of matrix reproducibly suppressed HDMEC proliferation and increased HDMEC adhesion to the substratum. These processes of matrix deposition and downregulation of endothelial cell proliferation, hallmarks of differentiating new capillaries in the end of angiogenesis, were recapitulated in our cell culture system by decreasing the matrix-degrading activity. These data suggest that our cell culture assay provides a simple and feasible model system for the study of capillary endothelial cell differentiation and vessel maturation in vitro.

scholarly journals The Damage Sensory Molecule TRPA1 Positively Regulates Endogenous Thymic Regeneration after Damage

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 67-67
Author(s):  
Kimon Argyropoulos ◽  
Enrico Velardi ◽  
Jennifer Tsai ◽  
Amina Lazrak ◽  
Lorenz Jahn ◽  
...  
Keyword(s):  
Lipid Peroxidation ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Epithelial Cells ◽  
Thymic Epithelial Cells ◽  
Lipid Peroxidation Products ◽  
Positive Effects ◽  
Damage Sensing ◽  
Cell Counts ◽  
Thymic Regeneration

Abstract The thymus is extremely sensitive to exogenous insults but has a remarkable capacity to regenerate which is lost with age. Reactive oxygen species (ROS) accumulate early after tissue damage and despite their toxic potential, ROS and their byproducts (such as lipid peroxidation products-LPPs) can act as regeneration signals by activating membrane or intracellular sensors and subsequent stress-response signalling pathways. Using Sublethal Total Body Irradiation (SL-TBI) as a model of acute thymic injury, we found a rapid accumulation of thymic ROS as well as lipid peroxidation products on cell membranes after SLTBI (Figure 1A&B). The damage-sensing ion channel Transient Receptor Potential cation channel family A member 1 (TRPA1) represents one of the major damage sensing receptors that can mediate cellular responses to oxidative stress mediators, such as LPPs. Using immunofluorescence (IF) microscopy we found that TRPA1 is enriched in the thymic medulla. Interestingly, although TRPA1 has been classically identified in nociceptive fibers, the major TRPA1 expressing structures in the thymus were not nerve fiber terminals, but primarily thymic endothelial cells (Figure1C), fibroblasts and subsets of epithelial cells. We have recently demonstrated that thymic endothelial cells can regulate regeneration through secretion of BMP-4, which can enhance Foxn1 expression and proliferation of thymic epithelial cells. In order to assess the functional role of TRPA1 in thymic regeneration after injury, we utilized TRPA1 knockout (TRPA1-/-) mice and quantified thymic reconstitution after SL-TBI. TRPA1-/- mice had significantly lower thymic cellularity compared to their age- and sex-matched WT controls, suggesting an association between TRPA1 deficiency and delayed endogenous thymic recovery (Figure 1D). The major deficit in thymocyte counts primarily affected double negative-4 (DN4), double positive (DP) and CD4+ single positive (SP-CD4+) thymocyte numbers. The thymic stroma of TRPA1-/- mice had lower endothelial cell and fibroblast counts (Figure 1D). In accordance with these findings drinking water administration of the TRPA1 agonist Allyl-Isothiocyanate (AITC), resulted in enhanced thymic regeneration after radiation exposure. Besides its positive effects on thymocyte counts, AITC significantly augmented endothelial cell counts after irradiation (Figure 1E). In conclusion these results suggest that TRPA1 plays a non-redundant role in thymic regeneration and that exogenous TRPA1 stimulation can enhance immune recovery after damage. Disclosures van den Brink: Seres: Research Funding; Jazz Pharmaceuticals: Consultancy; PureTech Health: Consultancy; Therakos Institute: Other: Speaking engagement.

scholarly journals Differential binding of hyaluronan on the surface of tissue-specific endothelial cell lines.

2008 ◽  
Vol 55 (1) ◽  
pp. 35-42 ◽  
Author(s):  
Karol Szczepanek ◽  
Claudine Kieda ◽  
Joanna Cichy
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
Tissue Specific ◽  
Independent Mechanism ◽  
Differential Binding ◽  
And Function ◽  
Pathologic Processes ◽  
Tissue Origin

Tissue-specific heterogeneity of endothelial cells, both structural and functional, plays a crucial role in physiologic as well as pathologic processes, including inflammation, autoimmune diseases and tumor metastasis. This heterogeneity primarily results from the differential expression of adhesion molecules that are involved in the interactions between endothelium and circulating immune cells or disseminating tumor cells. Among these molecules present on endothelial cells is hyaluronan (HA), a glycosaminoglycan that contributes to primary (rolling) interactions through binding to its main receptor CD44 expressed on leukocytes and tumor cells. While the regulation of CD44 expression and function on either leukocytes or tumor cells has been well characterized, much less is known about the ability of endothelial cells to express HA on their surface. Therefore, in these studies we analyzed HA levels on tissue-specific endothelium. We used endothelial cell lines of different origin, including lung, skin, gut and lymph nodes that had been established previously as model lines to study interactions between the endothelium and leukocytes/tumor cells. Our results indicate that HA is accumulated on the surface of all endothelial cells examined. Moreover, retention of endogenous HA differs between the lines and may depend on their tissue origin. Analysis of binding of exogenous HA reveals the presence of specific HA binding sites on all endothelial cell lines tested. However, the retention of endogenous HA and the binding of exogenous HA is mediated through a CD44-independent mechanism.

scholarly journals Cytokine-Mediated Downregulation of Coxsackievirus-Adenovirus Receptor in Endothelial Cells

2004 ◽  
Vol 78 (15) ◽  
pp. 8047-8058 ◽  
Author(s):  
Theresa Vincent ◽  
Ralf F. Pettersson ◽  
Ronald G. Crystal ◽  
Philip L. Leopold
Keyword(s):  
Endothelial Cells ◽  
Epithelial Cells ◽  
Protein Expression ◽  
Western Blot ◽  
Inflammatory Cytokines ◽  
Cell Physiology ◽  
Mrna Levels ◽  
Cytokine Treatment ◽  
Adenovirus Receptor ◽  
The Impact

ABSTRACT Endothelial cells have the ability to change their complement of cell surface proteins in response to inflammatory cytokines. We hypothesized that the expression of the coxsackievirus-adenovirus receptor (CAR), a viral receptor and putative cell-cell adhesion molecule, may be altered during the response of endothelial cells to inflammation. To test this hypothesis, we evaluated CAR protein and mRNA levels in human umbilical vein endothelial cells after they were exposed to tumor necrosis factor alpha, gamma interferon, or a combination of the two cytokines. Flow cytometric and Western blot analyses indicated that cytokine treatment led to a synergistic decrease in CAR protein expression. A Western blot analysis showed that CAR levels decreased to 16% ± 4% or 1% ± 4% of the CAR protein levels in untreated cells with either 24 or 48 h of cytokine treatment, respectively. Quantitative reverse transcription-PCR demonstrated that the combination treatment caused CAR mRNA levels to decrease to 21% ± 12% or 5% ± 3% of the levels in untreated cells after a 24- or 48-h cytokine treatment, respectively. Reduced CAR expression led to a decrease in adenovirus (Ad) binding of 80% ± 3% (compared with untreated endothelial cells), with a subsequent decrease in Ad-mediated gene transfer that was dependent on the dose and duration of cytokine treatment but not on the dose of Ad. A similar decrease in CAR protein level and susceptibility to Ad infection was observed in human microvascular endothelial cells, while CAR expression on normal human bronchial epithelial cells or A549 lung epithelial cells was less affected by cytokine treatments. Taken together, the data demonstrate that inflammatory cytokines decrease CAR mRNA and protein expression with a concomitant decrease in Ad binding, reflecting the impact of cell physiology on the function of CAR and the potential effect of inflammation on the ability of Ad to transfer genes to endothelial cells.

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