Abstract P262: Spike Protein 1 Of Sars-cov-2 Increases Interferon Stimulated Genes And Induces An Immune/inflammatory Responses In Human Endothelial Cells.

Hypertension ◽  
2021 ◽  
Vol 78 (Suppl_1) ◽  
Author(s):  
Francisco J Rios ◽  
Augusto C Montezano ◽  
Livia L Camargo ◽  
Rheure A Lopes ◽  
Eihu Aranday-Cortes ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Inflammatory Responses ◽  
Microvascular Endothelial Cells ◽  
Human Endothelial Cells ◽  
Angiotensin Converting Enzyme 2 ◽  
Interferon Stimulated Genes ◽  
Ifn Alpha ◽  
Enos Phosphorylation ◽  
Microvascular Endothelial ◽  
Transmembrane Serine Protease

Introduction: Interferon (IFN) alpha (IFNα) and lambda3 (IFNL3) constitute the first line of immunity against SARS-CoV-2 infection by increasing interferon-stimulated genes (ISGs). IFNs influence the expression of angiotensin-converting enzyme 2 (ACE2), the receptor for S-protein (S1P) of SARS-CoV-2. Here we hypothesized that in human microvascular endothelial cells (EC) IFNL3 and IFNα influence ACE2 and immune/inflammatory responses mediated by S1P. Methods: EC were stimulated with S1P of SARS-CoV-2 (1 μg/10^6 cells), IFNα (100 ng/mL) or IFNL3 (100 IU/mL). Because ACE2, metalloproteinase domain 17 (ADAM17) and type II transmembrane serine protease (TMPRSS2) are important for SARS-CoV-2 infection, cells were treated with inhibitors of ADAM17 (marimastat, 3.8nM and TAPI-1, 100nM), ACE2 (MLN4760, 440pM), and TMPRSS2 (camostat, 50μM). Expression of ISGs (ISG15, IFIT1, and MX1) was investigated by real-time PCR (5h) and protein expression by immunoblotting (24h). Results: EC stimulated with S1P increased expression of ISGs: ISG15 (2 fold), IFIT1 (6 fold), MX1 (6 fold) (n=12, p<0.05). EC exhibited higher responses to IFNα (ISG15: 16 fold, IFIT1: 21 fold, MX1: 31 fold) than to IFNL3 (ISG15: 1.7 fold, IFIT1: 1.9 fold, MX1: 1.7 fold) (p<0.05). S1P increased gene expression of IL-6 (1.3 fold), TNFα (6.2 fold) and IL-1β (3.3 fold), effects that were maximized 100% by IFNα. Only marimastat inhibited S1P effects. IL-6 was increased by IFNα (1230 pg/mL) and IFNL3 (1124 pg/mL) vs control (591pg/mL). IFNα increased expression of ACE2 (75 kDa) (63%), ADAM17 (36%), and TMPRSS2 (65%). This was associated with increased phosphorylation of Stat1 (134%), Stat2 (102%), ERK1/2 (42%). Nitric oxide production and eNOS phosphorylation (Ser1177) were reduced by IFNα and (40%) and IFNL3 (40%). Conclusions: In human microvascular endothelial cells, S1P, IFNα and IFNL3 induced an immune response characterized by increased expression of interferon-stimulated genes and IL-6 production, processes that involve ADAM17. Inflammation induced by S1P was amplified by IFNα. Our novel findings demonstrate that S1P induces an endothelial immune/inflammatory response that may be important in endotheliitis associated with COVID-19.

scholarly journals Induction of the S100 Chemotactic Protein, CP-10, in Murine Microvascular Endothelial Cells by Proinflammatory Stimuli

Blood ◽  
1997 ◽  
Vol 90 (12) ◽  
pp. 4812-4821 ◽  
Author(s):  
Tina Yen ◽  
Craig A. Harrison ◽  
Jannine M. Devery ◽  
Sharon Leong ◽  
Siiri E. Iismaa ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Inflammatory Responses ◽  
S100 Protein ◽  
Calcium Ionophore ◽  
Delayed Type Hypersensitivity ◽  
Microvascular Endothelial Cells ◽  
Mrna Induction ◽  
Chemotactic Protein ◽  
Microvascular Endothelial

Abstract Microvascular endothelial cells (EC) have multiple functions in inflammatory responses, including the production of chemoattractants that enhance leukocyte transmigration into tissues. Chemotactic protein, 10 kD (CP-10), is an S100 protein with potent chemotactic activity for myeloid cells in vitro and in vivo and is expressed in neutrophils and lipopolysaccharide (LPS)-activated macrophages. We show here that CP-10 is induced in murine endothelioma cell lines (bEnd-3, sEnd-1, and tEnd-1) after activation with LPS and interleukin-1 (IL-1) but not tumor necrosis factor α (TNFα) or interferon γ (IFNγ). Induction was not mediated by endogenous release of IL-1 or TNFα and was not directly upregulated by phorbol myristate acetate, calcium ionophore, or vitamin D3. EC were exquisitely sensitive to IL-1 activation (3.4 U/mL) and CP-10 mRNA induction with IL-1 occurred earlier (8 hours) than with LPS (12 hours). Furthermore, some microvessels and capillaries in delayed-type hypersensitivity lesions expressed cytoplasmic CP-10. Responses to LPS and not IL-1 in vitro were regulated by the degree of cell confluence and by TNFα costimulation. The related MRP-14 mRNA had a different induction pattern. Monomeric and homodimeric CP-10 upregulated by activation was predominantly cell-associated. EC-derived CP-10 may contribute to amplification of inflammatory processes by enhancing leukocyte shape changes and transmigration in the microcirculation.

scholarly journals Anaplasma phagocytophilum-Borrelia burgdorferi Coinfection Enhances Chemokine, Cytokine, and Matrix Metalloprotease Expression by Human Brain Microvascular Endothelial Cells

2007 ◽  
Vol 14 (11) ◽  
pp. 1420-1424 ◽  
Author(s):  
Dennis J. Grab ◽  
Elvis Nyarko ◽  
Nicole C. Barat ◽  
Olga V. Nikolskaia ◽  
J. Stephen Dumler
Keyword(s):  
Endothelial Cells ◽  
Borrelia Burgdorferi ◽  
Human Brain ◽  
Anaplasma Phagocytophilum ◽  
Inflammatory Responses ◽  
Matrix Metalloproteases ◽  
Microvascular Endothelial Cells ◽  
Brain Microvascular Endothelial Cells ◽  
Necrosis Factor Alpha ◽  
Microvascular Endothelial

ABSTRACT Borrelia burgdorferi and Anaplasma phagocytophilum coinfect and are transmitted by Ixodes species ticks. Clinical indicators suggest that A. phagocytophilum coinfection contributes to the severity, dissemination, and, possibly, sequelae of Lyme disease. Previous in vitro studies showed that spirochete penetration through human brain microvascular endothelial cells of the blood-brain barrier is facilitated by endothelial cell-derived matrix metalloproteases (MMPs). A. phagocytophilum-infected neutrophils continuously release MMPs and other vasoactive biomediators. We examined B. burgdorferi infection of brain microvascular barriers during A. phagocytophilum coinfection and showed that coinfection enhanced reductions in transendothelial electrical resistance and enhanced or synergistically increased production of MMPs (MMP-1, -3, -7, -8, and -9), cytokines (interleukin 6 [IL-6], IL-10, and tumor necrosis factor alpha), and chemokines (IL-8 and macrophage inflammatory protein 1α) known to affect vascular permeability and inflammatory responses.

scholarly journals Insulin and IGF1 receptors in human cardiac microvascular endothelial cells: metabolic, mitogenic and anti-inflammatory effects

2012 ◽  
Vol 215 (1) ◽  
pp. 89-96 ◽  
Author(s):  
Karolina Bäck ◽  
Rakibul Islam ◽  
Git S Johansson ◽  
Simona I Chisalita ◽  
Hans J Arnqvist
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
Inflammatory Responses ◽  
Receptor Protein ◽  
Microvascular Endothelial Cells ◽  
Tnf Α ◽  
Glucose Accumulation ◽  
Anti Inflammatory ◽  
Microvascular Endothelial ◽  
Cardiac Microvascular Endothelial Cells

Diabetes is associated with microcirculatory dysfunction and heart failure and changes in insulin and IGF1 levels. Whether human cardiac microvascular endothelial cells (HMVEC-Cs) are sensitive to insulin and/or IGF1 is not known. We studied the role of insulin receptors (IRs) and IGF1 receptors (IGF1Rs) in metabolic, mitogenic and anti-inflammatory responses to insulin and IGF1 in HMVEC-Cs and human umbilical vein endothelial cells (HUVECs). IR and IGF1R gene expression was studied using real-time RT-PCR. Receptor protein expression and phosphorylation were determined by western blot and ELISA. Metabolic and mitogenic effects were measured as glucose accumulation and thymidine incorporation. An E-selectin ELISA was used to investigate inflammatory responses. According to gene expression and protein in HMVEC-Cs and HUVECs, IGF1R is more abundant than IR. Immunoprecipitation with anti-IGF1R antibody and immunoblotting with anti-IR antibody and vice versa, showed insulin/IGF1 hybrid receptors in HMVEC-Cs. IGF1 at a concentration of 10−8 mol/l significantly stimulated phosphorylation of both IGF1R and IR in HMVEC-Cs. In HUVECs IGF1 10−8 mol/l phosphorylated IGF1R. IGF1 stimulated DNA synthesis at 10−8 mol/l and glucose accumulation at 10−7 mol/l in HMVEC-Cs. TNF-α dramatically increased E-selectin expression, but no inflammatory or anti-inflammatory effects of insulin, IGF1 or high glucose were seen. We conclude that HMVEC-Cs express more IGF1Rs than IRs, and mainly react to IGF1 due to the predominance of IGF1Rs and insulin/IGF1 hybrid receptors. TNF-α has a pronounced pro-inflammatory effect in HMVEC-Cs, which is not counteracted by insulin or IGF1.

Abstract 40: SARS-CoV-2/ACE2 Induces Vascular Inflammatory Responses In Human Microvascular Endothelial Cells Independently Of Viral Replication

Hypertension ◽  
2021 ◽  
Vol 78 (Suppl_1) ◽  
Author(s):  
Augusto C Montezano ◽  
Livia Camargo ◽  
Sheon Mary ◽  
Karla B Neves ◽  
Francisco J Rios ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Viral Replication ◽  
Viral Infection ◽  
Protein Identification ◽  
Inflammatory Responses ◽  
Vascular Inflammation ◽  
Plaque Assay ◽  
Microvascular Endothelial Cells ◽  
Label Free ◽  
Microvascular Endothelial

SARS-CoV-2, the virus responsible for COVID19, binds to ACE2, via its spike protein S1 subunit, leading to viral infection and respiratory disease. COVID-19 is associated with cardiovascular disease and systemic inflammation. Since ACE2 is expressed in vascular cells we questioned whether SARS-CoV-2 induces vascular inflammation and whether this is related to viral infection. Human microvascular endothelial cells (EC) were exposed to recombinant S1p (rS1p) 0.66 μg/mL for 10 min, 5h and 24h. Gene expression was assessed by RT-PCR and levels of IL6 and MCP1, as well as ACE2 activity, were assessed by ELISA. Expression of ICAM1 and PAI1 was assessed by immunoblotting. ACE2 activity was blocked by MLN4760 (ACE2 inhibitor) and siRNA. Viral infection was assessed by exposing Vero E6 (kidney epithelial cells; pos ctl) and EC to 10 5 pfu of SARS-CoV-2 where virus titre was measured by plaque assay. Co-IP coupled mass spectrometry protein identification and label free proteomics were used to investigate ACE2-mediated signalling. rS1p increased IL6 mRNA (14.2±2.1 vs. C:0.61±0.03 2^-ddCT) and levels (1221.2±18.3 vs. C:22.77±3.2 pg/mL); MCP1 mRNA (5.55±0.62 vs. C:0.65±0.04 2^-ddCT) and levels (1110±13.33 vs. C:876.9±33.4 pg/mL); ICAM1 (17.7±3.1 vs. C:3.9±0.4 AU) and PAI1 (5.6±0.7 vs. C: 2.9±0.2), p<0.05. MLN4760, but not rS1p, decreased ACE2 activity (367.4±18 vs. C: 1011±268 RFU, p<0.05) and blocked rS1p effects on ICAM1 and PAI1. ACE2 siRNA blocked rS1p-induced IL6 release, ICAM1, and PAI1 responses as well as rS1p-induced NFκB activation. Proteomics analysis of the global effect of rS1, identified biological process enrichment of proteins from virus transcription and NFκB signalling. ACE2 Co-IP identified 216 interacting proteins (filtered with ≥1 unique peptide, 1% FDR), linked to cytokine production and inflammation. EC were not susceptible to SARS-CoV-2 infection, while the virus replicated well in Vero E6. In conclusion, we demonstrate that rS1p induces an inflammatory response through ACE2 in endothelial cells. These effects seem to be independent of viral infection. Our findings suggest that vascular inflammation in COVID-19 involves activation of ACE2-mediated pro-inflammatory signalling that may be unrelated to viral replication.

scholarly journals Colquhounia Root Tablet Protects Rat Pulmonary Microvascular Endothelial Cells against TNF-α-Induced Injury by Upregulating the Expression of Tight Junction Proteins Claudin-5 and ZO-1

2018 ◽  
Vol 2018 ◽  
pp. 1-11 ◽  
Author(s):  
Wenjie Zhou ◽  
Guocui Shi ◽  
Jijia Bai ◽  
Shenmao Ma ◽  
Qinfu Liu ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Tight Junction ◽  
Cell Injury ◽  
Inflammatory Responses ◽  
Microvascular Endothelial Cells ◽  
Protective Effects ◽  
Tight Junction Proteins ◽  
Pulmonary Microvascular Endothelial Cells ◽  
Microvascular Endothelial ◽  
Junction Proteins

Background. There are currently limited effective pharmacotherapy agents for acute lung injury (ALI). Inflammatory response in the lungs is the main pathophysiological process of ALI. Our preliminary data have shown that colquhounia root tablet (CRT), a natural herbal medicine, alleviates the pulmonary inflammatory responses and edema in a rat model with oleic acid-induced ALI. However, the potential molecular action mechanisms underlining its protective effects against ALI are poorly understood. This study aimed to investigate the effects and mechanism of CRT in rat pulmonary microvascular endothelial cells (PMEC) with TNF-α-induced injury. Methods. PMECs were divided into 6 groups: normal control, TNF-α (10 ng/mL TNF-α), Dex (1×10-6 M Dex + 10 ng/mL TNF-α), CRT high (1000 ng/mL CRT + 10 ng/mL TNF-α), CRT medium (500 ng/mL CRT + 10 ng/mL TNF-α), and CRT low group (250 ng/mL CRT + 10 ng/mL TNF-α). Cell proliferation and apoptosis were detected by MTT assay and flow cytometry. Cell micromorphology was observed under transmission electron microscope. The localization and expression of tight junction proteins Claudin-5 and ZO-1 were analyzed by immunofluorescence staining and Western blot, respectively. Results. TNF-a had successfully induced an acute endothelial cell injury model. Dex and CRT treatments had significantly stimulated the growth and reduced the apoptosis of PMECs (all p < 0.05 or 0.01) and alleviated the TNF-α-induced cell injury. The expression of Claudin-5 and ZO-1 in Dex and all 3 CRT groups was markedly increased compared with TNF-a group (all p < 0.05 or 0.01). Conclusion. CRT effectively protects PMECs from TNF-α-induced injury, which might be mediated via stabilizing the structure of tight junction. CRT might be a promising, effective, and safe therapeutic agent for the treatment of ALI.

Lipopolysaccharide Induces the Antiapoptotic Molecules, A1 and A20, in Microvascular Endothelial Cells

Blood ◽  
1998 ◽  
Vol 92 (8) ◽  
pp. 2759-2765 ◽  
Author(s):  
Xiaolong Hu ◽  
Esther Yee ◽  
John M. Harlan ◽  
Fred Wong ◽  
Aly Karsan
Keyword(s):  
Endothelial Cells ◽  
Zinc Finger Protein ◽  
Microvascular Endothelial Cells ◽  
Human Endothelial Cells ◽  
Soluble Cd14 ◽  
Finger Protein ◽  
Microvascular Endothelial ◽  
American Society ◽  
Dependent Pathway ◽  
Necrosis Factor

The effect of lipopolysaccharide (LPS) on endothelial cells is a key component of the inflammatory response seen in Gram-negative sepsis. LPS does not cause death of cultured human endothelial cells. However, when the expression of new proteins is inhibited by cycloheximide, microvascular endothelial cells in culture undergo apoptosis. This finding suggests that LPS induces apoptotic and antiapoptotic pathways, with the antiapoptotic response being dependent on the synthesis of new proteins. Concurrent activation of apoptotic and antiapoptotic pathways has previously been documented for tumor necrosis factor (TNF). In the case of TNF, the antiapoptotic signal has been attributed to at least two cytoprotective proteins: the Bcl-2 homologue, A1, and the zinc-finger protein, A20. In this study, we demonstrate that both these molecules are induced in microvascular endothelial cells by LPS. Enforced overexpression of either A1 or A20 inhibits LPS and cycloheximide-initiated apoptosis. Induction of A1 and A20 does not require synthesis of intermediary proteins, but is dependent on the presence of soluble CD14. In addition, we show that inhibition of signaling by the transcription factor, NF-κB, blocks accumulation of A1 and A20 mRNA. Taken together, our findings suggest that LPS directly induces expression of the cytoprotective proteins, A1 and A20, via a CD14-dependent pathway requiring activation of NF-κB. © 1998 by The American Society of Hematology.

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