scholarly journals CD209L/L-SIGN and CD209/DC-SIGN act as receptors for SARS-CoV-2 and are differentially expressed in lung and kidney epithelial and endothelial cells

2020 ◽  
Author(s):  
Razie Amraei ◽  
Wenqing Yin ◽  
Marc A. Napoleon ◽  
Ellen L. Suder ◽  
Jacob Berrigan ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Vascular System ◽  
Virus Entry ◽  
Antiviral Drug ◽  
Cell Types ◽  
Host Cells ◽  
Human Endothelial Cells ◽  
High Importance ◽  
Lung And Kidney ◽  
Low Expression

AbstractAs the COVID-19 pandemic continues to spread, investigating the processes underlying the interactions between SARS-CoV-2 and its hosts is of high importance. Here, we report the identification of CD209L/L-SIGN and the related protein CD209/DC-SIGN as receptors capable of mediating SARS-CoV-2 entry into human cells. Immunofluorescence staining of human tissues revealed prominent expression of CD209L in the lung and kidney epithelium and endothelium. Multiple biochemical assays using a purified recombinant SARS-CoV-2 spike receptor binding domain (S-RBD) and ectopically expressed CD209L and CD209 revealed that CD209L and CD209 interact with S-RBD. CD209L contains two N-glycosylation sequons, at sites N92 and N361, but only site N92 is occupied. Removal of the N-glycosylation at this site enhances the binding of S-RBD with CD209L. CD209L also interacts with ACE2, suggesting a role for heterodimerization of CD209L and ACE2 in SARS-CoV-2 entry and infection in cell types where both are present. Furthermore, we demonstrate that human endothelial cells are permissive to SARS-CoV-2 infection and interference with CD209L activity by knockdown strategy or with soluble CD209L inhibits virus entry. Our observations demonstrate that CD209L and CD209 serve as alternative receptors for SARS-CoV-2 in disease-relevant cell types, including the vascular system. This property is particularly important in tissues where ACE2 has low expression or is absent, and may have implications for antiviral drug development.SignificanceUnderstanding the interactions between SARS-CoV-2 with host cells is of high importance. ACE2 is recognized as a major entry receptor, but SARS-CoV-2 may also employ alternative receptors for cell entry and these may hold the key to infection in tissues, where ACE2 has a low expression level or is absent. We identify CD209L/L-SIGN and CD209/DC-SIGN as receptors for SARS-CoV-2. We show that CD209L is N-glycosylated and this modification modulates the binding of CD209L with spike protein. CD209L interacts with ACE2, suggesting that CD209L and ACE2 could function as co-receptors for SARS-CoV-2 entry and infection. Human endothelial cells are permissive to SARS-CoV-2 infection. We show that interfering with CD209L activity in endothelial cells by knockdown or with introduction of soluble CD209L inhibits virus entry, suggesting a novel target for development of antiviral drugs.

scholarly journals HiPS-Endothelial Cells Acquire Cardiac Endothelial Phenotype in Co-culture With hiPS-Cardiomyocytes

2021 ◽  
Vol 9 ◽  
Author(s):  
Emmi Helle ◽  
Minna Ampuja ◽  
Alexandra Dainis ◽  
Laura Antola ◽  
Elina Temmes ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Animal Studies ◽  
Cardiac Development ◽  
Cell Types ◽  
Human Endothelial Cells ◽  
Bidirectional Communication ◽  
Induced Pluripotent ◽  
And Function ◽  
Cell Crosstalk

Cell-cell interactions are crucial for organ development and function. In the heart, endothelial cells engage in bidirectional communication with cardiomyocytes regulating cardiac development and growth. We aimed to elucidate the organotypic development of cardiac endothelial cells and cardiomyocyte and endothelial cell crosstalk using human induced pluripotent stem cells (hiPSC). Single-cell RNA sequencing was performed with hiPSC-derived cardiomyocytes (hiPS-CMs) and endothelial cells (hiPS-ECs) in mono- and co-culture. The presence of hiPS-CMs led to increased expression of transcripts related to vascular development and maturation, cardiac development, as well as cardiac endothelial cell and endocardium-specific genes in hiPS-ECs. Interestingly, co-culture induced the expression of cardiomyocyte myofibrillar genes and MYL7 and MYL4 protein expression was detected in hiPS-ECs. Major regulators of BMP- and Notch-signaling pathways were induced in both cell types in co-culture. These results reflect the findings from animal studies and extend them to human endothelial cells, demonstrating the importance of EC-CM interactions during development.

EXPRESSION OF PLATELET ALLOANTIGENS ON HUMAN ENDOTHELIAL CELLS AND HEL CELLS

1987 ◽  
Author(s):  
Y Kawai ◽  
R R Montgomery ◽  
K Furihata ◽  
T J Kunicki
Keyword(s):  
Endothelial Cells ◽  
Protein A ◽  
Cell Types ◽  
Membrane Glycoproteins ◽  
Human Endothelial Cells ◽  
Immunoblot Assay ◽  
Sds Page ◽  
Hel Cells ◽  
Erythroleukemia Cell ◽  
Different Cell Types

Analogs of platelet membrane glycoproteins IIb and IIIa (GPIIb-IIIa) have been shown to be synthesized and expressed by human endothelial cells (HEC), a human erythroleukemia cell line (HEL) and various other cells. Previous studies from our laboratory demonstrated that the platelet alloantigen P1A1, is expressed on HEC GPIIIa. Other alloantigen systems, namely, Pen and Bak, are known to be localized on platelet GPIIIa and GPIIb, respectively. Utilizing additional antibodies from patients with PTP specific for Pena, Baka, and Bakb allo-antigens, and isoantibodies (iso-ab) from a patient with Glanzmann's Thrombasthenia (GT), we have studied cultured HEC and HEL cells for expression of epitopes recognized by these antibodies. HEC and HEL cells were meta-bolically labeled with 35S-methionine and lysed in 0.5% TX-100 in the presence of 5mM EDTA. Soluble antigens were immunoprecipitated with these antibodies coupled to Protein A-Sepharose and subjected to SDS-PAGE and fluorography. Anti-Pena and the GT iso-ab reacted with the GPIIb-IIIa complex from both HEC and HEL lysates, but anti-Baka and anti-Bakb failed to immunoprecipitate GPIIb-IIIa analogs from either HEC or HEL. In an immunoblot assay, the GT iso-ab bound to GPIIIa of both HEC and HEL. Anti-Pena failed to react with SDS-denatured proteins. HEL GPIIIa migrates slightly faster than HEC GPIIIa and slightly slower than platelet GPIIIa. These results indicate that the epitopes of platelet GPIIIa recognized by alloantibodies and isoantibodies are shared by GPIIIa analogs of HEC and HEL. GPIIb-associated alloantigens are not expressed by HEC and HEL GPIIb analogs, an observation that is consistent with the decreased structural homology between GPIIb analogs derived from different cell types.

scholarly journals Attachment of cultured human endothelial cells is promoted by specific association with S protein (vitronectin) as well as with the ternary S protein-thrombin-antithrombin III complex

Blood ◽  
1988 ◽  
Vol 71 (6) ◽  
pp. 1581-1589 ◽  
Author(s):  
KT Preissner ◽  
E Anders ◽  
J Grulich-Henn ◽  
G Muller-Berghaus
Keyword(s):  
Endothelial Cells ◽  
Antithrombin Iii ◽  
Cell Attachment ◽  
Umbilical Vein ◽  
Cell Types ◽  
Human Endothelial Cells ◽  
S Protein ◽  
Major Vessel ◽  
Specific Association ◽  
Dose Dependent

Abstract The interaction of the multifunctional S protein (vitronectin) with cultured human endothelial cells of macrovascular and microvascular origin was investigated. Purified S protein, coated on polystyrene Petri dishes, induced dose-dependent and time-dependent attachment and spreading of human umbilical vein endothelial cells (HUVECs) as well as human omental tissue microvascular endothelial cells (HOTMECs) at 37 degrees C. Not only isolated S protein, but also the ternary S protein- thrombin-antithrombin III (STAT) complex promoted attachment of approximately 90% of the cells within 2 hours at an S protein concentration of 0.13 mumol/L. Inhibition of attachment in these experiments was achieved by the addition of the cell-attachment pentapeptide Gly-Arg-Gly-Asp-Ser and by monospecific antibodies against S protein, whereas nonrelated peptides or antibodies against fibronectin, fibrinogen, or von Willebrand factor (vWF) were ineffective. Direct binding of S protein to HUVECs and HOTMECs was studied with cells in suspension at a density of 1 x 10(6) cells/mL and was maximal after 120 minutes. S protein bound to both cell types in a dose-dependent fashion with an estimated dissociation constant Kd = 0.2 mumol/L. At a 200-fold to 500-fold molar excess of unlabeled S protein, greater than 80% of bound radiolabeled S protein was displaceable, whereas binding was reduced to 30% to 50% by addition of the pentapeptide, the STAT complex, or by physiologic concentrations of fibrinogen or vWF as well as Fab fragments of anti(human S protein)IgG, but not by Fab rabbit IgG. These findings present evidence for the specific association of S protein with endothelial cells ultimately leading to attachment and spreading of cells. Moreover, a novel function for the ternary STAT complex, which induced endothelial cell attachment and spreading virtually identical to free S protein, is described. These data further suggest a possible role for S protein during coagulation as major vessel wall-related adhesive protein at sites of vascular injury.

Expression of the Drug-Dependent Antigen for Quinine-Dependent Antiplatelet Antibodies on GP III a but not that on GPI b, II b or IX on Human Endothelial Cells

1990 ◽  
Vol 63 (02) ◽  
pp. 279-281 ◽  
Author(s):  
Sharron L Pfueller ◽  
Robyn A Bilston ◽  
Stephen Jane ◽  
Jannine Gibson
Keyword(s):  
Endothelial Cells ◽  
Cell Types ◽  
Dependent Manner ◽  
Igg Antibodies ◽  
Human Endothelial Cells ◽  
Thrombocytopenic Purpura ◽  
Antiplatelet Antibodies ◽  
Igg Binding ◽  
Drug Dependent ◽  
Surface Glycoproteins

SummaryIn quinine- and quinidine-induced thrombocytopenic purpura IgG antibodies are known to react in a drug-dependent manner with different combinations of surface glycoproteins (GP) Ib , IIb, IIIa and IX. Because endothelial cells share a number of properties of platelets, including the presence of GP Ilia and GPIb-like proteins, we have compared these two cell types for their quinine-dependent IgG binding abilities. By immunoblotting of endothelial cells, quinine-dependent IgG binding from four patient sera was observed only to a 93 kDa component corresponding to GP Ilia. Strong IgG binding independent of the drug was found at 170–180 kDa. Thus endothelial cells express the GP Ilia quinine-dependent epitope on platelet GP IIIa , but not those on other platelet glycoproteins.

scholarly journals The Human Cytomegalovirus Ribonucleotide Reductase Homolog UL45 Is Dispensable for Growth in Endothelial Cells, as Determined by a BAC-Cloned Clinical Isolate of Human Cytomegalovirus with Preserved Wild-Type Characteristics

2002 ◽  
Vol 76 (18) ◽  
pp. 9551-9555 ◽  
Author(s):  
Gabriele Hahn ◽  
Hanna Khan ◽  
Fausto Baldanti ◽  
Ulrich H. Koszinowski ◽  
M. Grazia Revello ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Clinical Isolate ◽  
Human Cytomegalovirus ◽  
Ribonucleotide Reductase ◽  
Human Fibroblasts ◽  
Artificial Chromosome ◽  
Cell Types ◽  
Factor X ◽  
Wild Type ◽  
Human Endothelial Cells

ABSTRACT An endothelial cell-tropic and leukotropic human cytomegalovirus (HCMV) clinical isolate was cloned as a fusion-inducing factor X-bacterial artificial chromosome in Escherichia coli, and the ribonucleotide reductase homolog UL45 was deleted. Reconstituted virus RVFIX and RVΔUL45 grew equally well in human fibroblasts and human endothelial cells. Thus, UL45 is dispensable for growth of HCMV in both cell types.

scholarly journals Human endothelial cells and fibroblasts express and produce the coagulation proteins necessary for thrombin generation

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Clay T. Cohen ◽  
Nancy A. Turner ◽  
Joel L. Moake
Keyword(s):  
Endothelial Cells ◽  
Thrombin Generation ◽  
Coagulation Factors ◽  
Cell Types ◽  
Human Endothelial Cells ◽  
Surface Binding ◽  
Prothrombinase Complex ◽  
Coagulation Proteins ◽  
Exogenous Addition ◽  
Activate Cell

AbstractIn a previous study, we reported that human endothelial cells (ECs) express and produce their own coagulation factors (F) that can activate cell surface FX without the additions of external proteins or phospholipids. We now describe experiments that detail the expression and production in ECs and fibroblasts of the clotting proteins necessary for formation of active prothrombinase (FV–FX) complexes to produce thrombin on EC and fibroblast surfaces. EC and fibroblast thrombin generation was identified by measuring: thrombin activity; thrombin–antithrombin complexes; and the prothrombin fragment 1.2 (PF1.2), which is produced by the prothrombinase cleavage of prothrombin (FII) to thrombin. In ECs, the prothrombinase complex uses surface-attached FV and γ-carboxyl-glutamate residues of FX and FII to attach to EC surfaces. FV is also on fibroblast surfaces; however, lower fibroblast expression of the gene for γ-glutamyl carboxylase (GGCX) results in production of vitamin K-dependent coagulation proteins (FII and FX) with reduced surface binding. This is evident by the minimal surface binding of PF1.2, following FII activation, of fibroblasts compared to ECs. We conclude that human ECs and fibroblasts both generate thrombin without exogenous addition of coagulation proteins or phospholipids. The two cell types assemble distinct forms of prothrombinase to generate thrombin.

scholarly journals Human Endothelial Cells And Fibroblasts Express And Produce The Coagulation Proteins Necessary For Thrombin Generation

2021 ◽  
Author(s):  
Clay Cohen ◽  
Nancy Turner ◽  
Joel Moake
Keyword(s):  
Endothelial Cells ◽  
Thrombin Generation ◽  
Coagulation Factors ◽  
Cell Types ◽  
Human Endothelial Cells ◽  
Surface Binding ◽  
Prothrombinase Complex ◽  
Coagulation Proteins ◽  
Exogenous Addition ◽  
Activate Cell

Abstract In a previous study, we reported that human endothelial cells (ECs) express and produce their own coagulation factors (F) that can activate cell surface FX without the additions of external proteins or phospholipids. We now describe experiments that detail the expression and production in ECs and fibroblasts of the clotting proteins necessary for formation of active prothrombinase (FV-FX) complexes to produce thrombin on EC and fibroblast surfaces. EC and fibroblast thrombin generation was identified by measuring: thrombin activity; thrombin-antithrombin complexes; and the prothrombin fragment 1.2 (PF1.2), which is produced by the prothrombinase cleavage of prothrombin (FII) to thrombin. In ECs, the prothrombinase complex uses surface-attached FV and g-carboxyl-glutamate residues of FX and FII to attach to EC surfaces. FV is also on fibroblast surfaces; however, lower fibroblast expression of the gene for γ-glutamyl carboxylase (GGCX) results in production of vitamin K-dependent coagulation proteins (FII and FX) with reduced surface binding. This is evident by the minimal surface binding of PF1.2, following FII activation, of fibroblasts compared to ECs. We conclude that human ECs and fibroblasts both generate thrombin without exogenous addition of coagulation proteins or phospholipids. The two cell types assemble distinct forms of prothrombinase to generate thrombin.

scholarly journals Staphylococcus aureus α-toxin induces acid sphingomyelinase release from a human endothelial cell line

2021 ◽  
Author(s):  
David Krones ◽  
Marcel Rühling ◽  
Katrin Anne Becker ◽  
Tobias C. Kunz ◽  
Carolin Sehl ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Endothelial Cells ◽  
Plasma Membrane ◽  
Extracellular Fluid ◽  
Fluid Phase ◽  
Cell Types ◽  
Acid Sphingomyelinase ◽  
Host Cells ◽  
Membrane Repair ◽  
Membrane Area

AbstractStaphylococcus aureus (S. aureus) is well known to express a plethora of toxins of which the pore-forming hemolysin A (α-toxin) is the best-studied cytolysin. Pore-forming toxins (PFT) permeabilize host membranes during infection thereby causing concentration-dependent effects in host cell membranes ranging from disordered ion fluxes to cytolysis. Host cells possess defense mechanisms against PFT attack, resulting in endocytosis of the breached membrane area and delivery of repair vesicles to the insulted plasma membrane as well as a concurrent release of membrane repair enzymes. Since PFTs from several pathogens have been shown to recruit membrane repair components, we here investigated whether staphylococcal α-toxin is able to induce these mechanisms in endothelial cells. We show that S. aureus α-toxin induced increase in cytosolic Ca2+ in endothelial cells, which was accompanied by p38 MAPK phosphorylation. Toxin challenge led to increased endocytosis of an extracellular fluid phase marker as well as increased externalization of LAMP1-positive membranes suggesting that peripheral lysosomes are recruited to the insulted plasma membrane. We further observed that thereby the lysosomal protein acid sphingomyelinase (ASM) was released into the cell culture medium. Thus, our results show that staphylococcal α-toxin triggers mechanisms in endothelial cells, which have been implicated in membrane repair after damage of other cell types by different toxins.

Either exogenous or endogenous fibronectin can promote adherence of human endothelial cells

1986 ◽  
Vol 82 (1) ◽  
pp. 263-280
Author(s):  
R.A. Clark ◽  
J.M. Folkvord ◽  
L.D. Nielsen
Keyword(s):  
Endothelial Cells ◽  
Wound Repair ◽  
Cell Types ◽  
Culture Conditions ◽  
Collagen Type Iv ◽  
Human Endothelial Cells ◽  
Small Vessels ◽  
Microvascular Cells

Recently, we have presented evidence that proliferating blood vessels produce and deposit fibronectin in situ during the angiogenesis of wound repair. This report extends these observations by demonstrating that human endothelial cells from both large and small vessels depend on fibronectin for their adherence in vitro. Endothelial cells were grown from human umbilical veins (HUVEC) by the method of Gimbrone and from the microvasculature of human omentum by the method of Kern, Knedler and Eckel. Second-passage cells were plated into microtitre wells that had been coated with 100 micrograms ml-1 of fibronectin, types I and III collagen, type IV collagen or laminin. After a 3-h incubation, adherent cells were solubilized with Zap-Isoton and quantified on a Coulter Counter. Under normal culture conditions HUVEC showed no preference for fibronectin substrates while microvascular cells always demonstrated a striking preference for fibronectin substrates. However, when HUVEC were exposed to 2.5 or 25 micrograms ml-1 of cycloheximide for 4 h before and during the adherence assays, the adherence to fibronectin was 50–200% greater than to types I and III collagen. Immunofluorescence studies showed that while HUVEC expressed a large quantity of surface fibronectin, microvascular cells expressed very little. Metabolic labelling studies confirmed that HUVEC cultures had substantial quantities of fibronectin in their cell layer while microvascular cells did not. In antibody blocking experiments, preincubation of fibronectin-coated surfaces with anti-fibronectin antibodies totally blocked microvascular cell adhesion but only abrogated HUVEC adherence by 50%, presumably since these latter cells were able to deposit additional fibronectin onto the surface during the 3 h assay period. In the presence of cycloheximide anti-fibronectin antibodies totally blocked HUVEC adherence. These results demonstrate that both endothelial cell types rely, at least in part, on fibronectin for adherence in vitro. HUVEC can synthesize, secrete and deposit enough fibronectin for their adherence in vitro, while microvascular cells rely on an exogenous source of fibronectin under these culture conditions. Thus, the increased blood vessel fibronectin observed during angiogenesis in vivo may mediate adherence of the proliferating and migrating endothelial cells.

scholarly journals Relative Roles of Listeriolysin O, InlA, and InlB inListeria monocytogenesUptake by Host Cells

2018 ◽  
Vol 86 (10) ◽  
Author(s):  
Christopher C. Phelps ◽  
Stephen Vadia ◽  
Eusondia Arnett ◽  
Yubo Tan ◽  
Xiaoli Zhang ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Host Cell ◽  
Cell Types ◽  
Bacterial Attachment ◽  
Host Cells ◽  
Listeriolysin O ◽  
Gene Encoding ◽  
Content Type ◽  
Membrane Perforation ◽  
Genes Encoding

ABSTRACTListeria monocytogenesis a facultative intracellular pathogen that infects a wide variety of cells, causing the life-threatening disease listeriosis.L. monocytogenesvirulence factors include two surface invasins, InlA and InlB, known to promote bacterial uptake by host cells, and the secreted pore-forming toxin listeriolysin O (LLO), which disrupts the phagosome to allow bacterial proliferation in the cytosol. In addition, plasma membrane perforation by LLO has been shown to facilitateL. monocytogenesinternalization into epithelial cells. In this work, we tested the host cell range and importance of LLO-mediatedL. monocytogenesinternalization relative to the canonical invasins, InlA and InlB. We measured the efficiencies ofL. monocytogenesassociation with and internalization into several human cell types (hepatocytes, cytotrophoblasts, and endothelial cells) using wild-type bacteria and isogenic single, double, and triple deletion mutants for the genes encoding InlA, InlB and LLO. No role for InlB was detected in any tested cells unless the InlB expression level was substantially enhanced, which was achieved by introducing a mutation (prfA*) in the gene encoding the transcription factor PrfA. In contrast, InlA and LLO were the most critical invasion factors, although they act in a different manner and in a cell-type-dependent fashion. As expected, InlA facilitates both bacterial attachment and internalization in cells that express its receptor, E-cadherin. LLO promotesL. monocytogenesinternalization into hepatocytes, but not into cytotrophoblasts and endothelial cells. Finally, LLO and InlA cooperate to increase the efficiency of host cell invasion byL. monocytogenes.

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