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 Lack of evidence of ACE2 expression and replicative infection by SARS-CoV-2 in human endothelial cells

2020 ◽  
Author(s):  
Ian McCracken ◽  
Gaye Saginc ◽  
Liqun He ◽  
Alik Huseynov ◽  
Alison Daniels ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Immune Cells ◽  
Vascular Endothelium ◽  
Productive Infection ◽  
Human Endothelial Cells ◽  
Transcriptomic Data ◽  
Small Vessels ◽  
Multiple Organs

AbstractA striking feature of severe COVID-19 is thrombosis in large as well as small vessels of multiple organs. This has led to the assumption that SARS-CoV-2 virus directly infects and damages the vascular endothelium. However, endothelial expression of ACE2, the cellular receptor for SARS-CoV-2, has not been convincingly demonstrated. Interrogating human bulk and single-cell transcriptomic data, we found ACE2 expression in endothelial cells to be extremely low or absent in vivo and not upregulated by exposure to inflammatory agents in vitro. Also, the endothelial chromatin landscape at the ACE2 locus showed presence of repressive and absence of activation marks, suggesting that the gene is inactive in endothelial cells. Finally, we failed to achieve infection and replication of SARS-CoV-2 in cultured human endothelial cells, which were permissive to productive infection by coronavirus 229E that uses CD13 as the receptor. Our data suggest that SARS-Cov-2 is unlikely to infect endothelial cells directly; these findings are consistent with a scenario where endothelial injury is indirectly caused by the infection of neighbouring epithelial cells and/or due to systemic effects mediated by immune cells, platelets, complement activation, and/or proinflammatory cytokines.

Abstract 897: Alliance of Blood Derived Endothelial Cells and Adipose Stromal Cells in Human Vasculogenesis

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Dmitry O Traktuev ◽  
Daniel N Prater ◽  
Aravind R Sanjeevaiah ◽  
Stephanie Merfeld-Clauss ◽  
Brian H Johnstone ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Stromal Cells ◽  
De Novo ◽  
Functional Integration ◽  
Cell Types ◽  
Specific Marker ◽  
Pronounced Difference ◽  
Adipose Stromal Cells ◽  
Small Vessels

Introduction Both Endothelial progenitor cells (EPC) and adipose stromal cells (ASC) are under investigation as therapies for cardiovascular diseases. Both cell types are capable of modulating vascular assembly and are, thereby, capable of directly promoting revascularization of ischemic tissues. We have shown that EPC differentiate into endothelial cells to form small vessels, whereas ASC have pericytic properties and naturally stabilize vessels. In this study we tested the possibility that ASC would interact with EPC to assemble de novo vessels in collagen in an in vivo chimeric implant. Methods and Results Collagen implants embedded with either umbilical cord blood EPC or adult ASC or a 4:1 mixture of both (2x10 6 cells/ml) were implanted subcutaneously into NOD/SCID mice. After 14 d implants were harvested and evaluated by immunohistochemistry. There was a pronounced difference among the groups in vascular network assembly. The majority of vessels formed in the EPC and ASC monocultures were small capillaries bounded by a single endothelial layer. Conversely, 100% of the plugs embedded with both cell types were highly invaded with multilayered arteriolar vessels. The density of the CD31 + vessels in the EPC and co-culture plugs was 26.6 ± 5.8 and 122.4 ± 9.8 per mm 2 , respectively. No CD31 + cells of human origin were detected in the ASC monocultures, indicating that ASC, which do not express this EC-specific marker, engage murine EC or form pseudovessels in this system. The density of α-SMA + vessels with lumens per mm 2 was 13.1 ± 3.6 (EPC), 10.2 ± 3.5 (ASC) and 124.7 ± 19.7 (co-culture). The total overlap of CD31 + and SMA + vessels demonstrates that mature, multilayered conduits were formed with the co-culture. Moreover, the majority of these vessels were filled with erythrocytes (92.5 ± 16.2 per mm 2 ), indicating inosculation with the native vasculature, which was confirmed by ultrasound with echogenic microbubbles and persisted to at least 4 months. Conclusion This study is the first to demonstrate that non-transformed human EPC and ASC cooperatively form mature and stable vasculature with subsequent functional integration into a host vasculature system.

Venular endothelial cells from bovine heart

1988 ◽  
Vol 254 (6) ◽  
pp. H1211-H1217 ◽  
Author(s):  
M. E. Schelling ◽  
C. J. Meininger ◽  
J. R. Hawker ◽  
H. J. Granger
Keyword(s):  
Endothelial Cells ◽  
In Vitro Model ◽  
Protein Transport ◽  
Culture Conditions ◽  
Support Cell ◽  
Vitro Model ◽  
Microvascular Cells ◽  
Selection Of ◽  
Coronary Angiogenesis

Coronary venular endothelial cells were isolated by a bead-perfusion technique that allowed the selection of endothelial cells from venules of a specific size. Culture conditions for the microvascular cells were established. Cells grew well in supplemented Dulbecco's modified Eagle's medium. The effect of various substrata on the proliferation of the venular endothelial cells was determined. Matrigel, gelatin, and fibronectin supported high levels of proliferation. Cell shape was correlated with ability of the substratum to support cell proliferation. Cells exhibiting a broad, flattened morphology achieved high levels of proliferation. The formation of vessel meshworks by the coronary venular endothelial cells provides an in vitro model for the study of coronary angiogenesis. Confluent monolayers of these cells can be utilized to examine mechanisms of water and protein transport across coronary venules.

scholarly journals Modulating the Substrate Stiffness to Manipulate Differentiation of Resident Liver Stem Cells and to Improve the Differentiation State of Hepatocytes

2016 ◽  
Vol 2016 ◽  
pp. 1-12 ◽  
Author(s):  
Angela Maria Cozzolino ◽  
Valeria Noce ◽  
Cecilia Battistelli ◽  
Alessandra Marchetti ◽  
Germana Grassi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Culture Method ◽  
Cell Types ◽  
Culture Conditions ◽  
Precursor Cells ◽  
Substrate Stiffness ◽  
Growth And Survival ◽  
Liver Stem Cells

In many cell types, several cellular processes, such as differentiation of stem/precursor cells, maintenance of differentiated phenotype, motility, adhesion, growth, and survival, strictly depend on the stiffness of extracellular matrix that,in vivo, characterizes their correspondent organ and tissue. In the liver, the stromal rigidity is essential to obtain the correct organ physiology whereas any alteration causes liver cell dysfunctions. The rigidity of the substrate is an element no longer negligible for the cultivation of several cell types, so that many data so far obtained, where cells have been cultured on plastic, could be revised. Regarding liver cells, standard culture conditions lead to the dedifferentiation of primary hepatocytes, transdifferentiation of stellate cells into myofibroblasts, and loss of fenestration of sinusoidal endothelium. Furthermore, standard cultivation of liver stem/precursor cells impedes an efficient execution of the epithelial/hepatocyte differentiation program, leading to the expansion of a cell population expressing only partially liver functions and products. Overcoming these limitations is mandatory for any approach of liver tissue engineering. Here we propose cell lines asin vitromodels of liver stem cells and hepatocytes and an innovative culture method that takes into account the substrate stiffness to obtain, respectively, a rapid and efficient differentiation process and the maintenance of the fully differentiated phenotype.

scholarly journals Robust and Scalable Angiogenesis Assay of Perfused 3D Human iPSC-Derived Endothelium for Anti-Angiogenic Drug Screening

2020 ◽  
Vol 21 (13) ◽  
pp. 4804
Author(s):  
Vincent van Duinen ◽  
Wendy Stam ◽  
Eva Mulder ◽  
Farbod Famili ◽  
Arie Reijerkerk ◽  
...  
Keyword(s):  
Drug Screening ◽  
Cell Formation ◽  
Cell Types ◽  
Culture Conditions ◽  
In Vitro Assays ◽  
Vegf Receptor ◽  
Screening Assay ◽  
Angiogenesis Assay

To advance pre-clinical vascular drug research, in vitro assays are needed that closely mimic the process of angiogenesis in vivo. Such assays should combine physiological relevant culture conditions with robustness and scalability to enable drug screening. We developed a perfused 3D angiogenesis assay that includes endothelial cells (ECs) from induced pluripotent stem cells (iPSC) and assessed its performance and suitability for anti-angiogenic drug screening. Angiogenic sprouting was compared with primary ECs and showed that the microvessels from iPSC-EC exhibit similar sprouting behavior, including tip cell formation, directional sprouting and lumen formation. Inhibition with sunitinib, a clinically used vascular endothelial growth factor (VEGF) receptor type 2 inhibitor, and 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO), a transient glycolysis inhibitor, both significantly reduced the sprouting of both iPSC-ECs and primary ECs, supporting that both cell types show VEGF gradient-driven angiogenic sprouting. The assay performance was quantified for sunitinib, yielding a minimal signal window of 11 and Z-factor of at least 0.75, both meeting the criteria to be used as screening assay. In conclusion, we have developed a robust and scalable assay that includes physiological relevant culture conditions and is amenable to screening of anti-angiogenic compounds.

scholarly journals Cerebromicrovascular endothelial cells are resistant to l-glutamate

2008 ◽  
Vol 295 (4) ◽  
pp. R1099-R1108 ◽  
Author(s):  
Ferenc Domoki ◽  
Béla Kis ◽  
Tamás Gáspár ◽  
Ferenc Bari ◽  
David W. Busija
Keyword(s):  
Endothelial Cells ◽  
Blood Brain Barrier ◽  
Primary Cultures ◽  
Glucose Deprivation ◽  
Brain Barrier ◽  
Cranial Window ◽  
Blood Brain ◽  
Microvascular Cells

Cerebral microvascular endothelial cells (CMVECs) have recently been implicated as targets of excitotoxic injury by l-glutamate (l-glut) or N-methyl-d-aspartate (NMDA) in vitro. However, high levels of l-glut do not compromise the function of the blood-brain barrier in vivo. We sought to determine whether primary cultures of rat and piglet CMVECs or cerebral microvascular pericytes (CMVPCs) are indeed sensitive to l-glut or NMDA. Viability was unaffected by 8-h exposure to 1–10 mM l-glut or NMDA in CMVECs or CMVPCs isolated from both species. Furthermore, neither 1 mM l-glut nor NMDA augmented cell death induced by 12-h oxygen-glucose deprivation in rat CMVECs or by 8-h medium withdrawal in CMVPCs. Additionally, transendothelial electrical resistance of rat CMVEC-astrocyte cocultures or piglet CMVEC cultures were not compromised by up to 24-h exposure to 1 mM l-glut or NMDA. The Ca2+ ionophore calcimycin (5 μM), but not l-glut (1 mM), increased intracellular Ca2+ levels in rat CMVECs and CMVPCs assessed with fluo-4 AM fluorescence and confocal microscopy. CMVEC-dependent pial arteriolar vasodilation to hypercapnia and bradykinin was unaffected by intracarotid infusion of l-glut in anesthetized piglets by closed cranial window/intravital microscopy. We conclude that cerebral microvascular cells are insensitive and resistant to glutamatergic stimuli in accordance with their in vivo role as regulators of potentially neurotoxic amino acids across the blood-brain barrier.

Controlled Adipose-derived Stromal Cells Differentiation into Adipose and Endothelial Cells in a 3D Structure Established by Cell-assembly Technique

2009 ◽  
Vol 24 (1_suppl) ◽  
pp. 31-47 ◽  
Author(s):  
Mingen Xu ◽  
Yongnian Van ◽  
Haixia Liu ◽  
Rui Yag ◽  
Xiaohong Wang
Keyword(s):  
Endothelial Cells ◽  
3D Structure ◽  
Spherical Shape ◽  
Cell Types ◽  
Tissue Growth ◽  
Cell Assembly ◽  
Adipose Tissue Engineering ◽  
Assembly Technique

One of the major obstacles in engineering thick and complex tissues while vascularizing tissues in vitro is to maintain cell viability during tissue growth and structural organization. Adipose-derived stromal (ADS) cells were used to establish a multicellular system through a cell-assembly technique. Attempts were made to control ADS cells differentiation into different targeted cell types according to their positions within an orderly 3D structure. Oil red 0 staining confirmed that the ADS cells in the structure differentiated into adipocytes with a spherical shape while immunostaining tests confirmed that the endothelial growth factor induced ADS cells on the walls of channels differentiated into mature endothelial cells and then organized into tubular structures throughout the engineered 3D structure. The endothelin-1 and nitric oxide release rules of the endothelial cells were coincidental with those in vivo. This study provides a new approach to engineer orderly endothelial vessel networks in vitro and has potential applications in adipose-tissue engineering.

scholarly journals Annexin 5 as a potential regulator of annexin 1 phosphorylation by protein kinase C. In vitro inhibition compared with quantitative data on annexin distribution in human endothelial cells

1993 ◽  
Vol 292 (3) ◽  
pp. 759-765 ◽  
Author(s):  
P Raynal ◽  
F Hullin ◽  
J M F Ragab-Thomas ◽  
J Fauvel ◽  
H Chap
Keyword(s):  
Endothelial Cells ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Inhibitory Effect ◽  
Human Endothelial Cells ◽  
Annexin 1 ◽  
Kinase C ◽  
Vitro Effect

In vitro phosphorylation of annexin 1 by purified rat brain protein kinase C (PKC) has been studied in the presence of annexin 5, which is not a substrate for PKC. Annexin 5 promoted a dose-dependent inhibition of annexin 1 phosphorylation, which could be overcome by increasing the concentration of phosphatidylserine (PtdSer). In addition, a close relationship was found between the amount of PtdSer uncovered by annexin 5 and the residual phosphorylation of annexin 1. These data fit with the ‘surface depletion model’ explaining the antiphospholipase activity of annexins. In order to check the possibility that the in vitro effect of annexin 5 could be of some physiological relevance, annexins 1, 2, and 5, as well as the light chain of calpactin 1 (p11), have been quantified in human endothelial cells by measuring the radioactivity bound to the proteins after Western blotting with specific antibodies and 125I-labelled secondary antibody. Our data indicate that annexins 1 and 5, PKC and PtdSer are present in human endothelial cells in relative amounts very similar to those used in vitro under conditions permitting the detection of the inhibitory effect of annexin 5. Since annexin 1 remained refractory to PKC-dependent phosphorylation in intact cells, we suggest that annexin 5 might exert its inhibitory effect towards PKC in vivo, provided that its binding to phospholipids can occur at physiological (micromolar) concentrations of Ca2+. This was previously shown to occur in vitro using phosphatidylethanolamine/phosphatidic acid vesicles [Blackwood and Ernst (1990) Biochem. J. 266, 195-200]. Using identical assay conditions, which also allowed expression of PKC activity, annexin 5 again inhibited annexin 1 phosphorylation without interfering with PKC autophosphorylation. These data suggest that annexins 1 and 5 might interact with each other on the lipid surface, resulting in a specific inhibition of annexin 1 phosphorylation by PKC. Whether a similar mechanism also occurs in vivo remains to be determined.

Role of inducible nitric oxide synthase in the regulation of leucocyte recruitment

2000 ◽  
Vol 100 (1) ◽  
pp. 1-12 ◽  
Author(s):  
Michael J. HICKEY
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Nitric Oxide Synthase ◽  
Cell Types ◽  
Adhesive Interactions ◽  
Oxide Synthase ◽  
Leucocyte Recruitment

Constitutively produced nitric oxide released by endothelial cells has been shown to act as an endogenous agent which inhibits the rolling and adhesion of leucocytes in the microcirculation. However, during various types of inflammation, expression of the inducible form of nitric oxide synthase (iNOS) can dramatically increase the amount of nitric oxide present in tissues. Furthermore, as iNOS can be expressed by a wide variety of cell types, the distribution of nitric oxide is likely to be altered relative to that in unstimulated tissue. Under these conditions, it is less well understood whether iNOS-derived nitric oxide retains the anti-adhesive capabilities of constitutively produced nitric oxide. This review summarizes work done to examine this issue. Three main approaches have been used. In vitro studies have examined the role of iNOS in adhesive interactions between stimulated endothelial cells and leucocytes, providing evidence of an anti-adhesive effect of iNOS. In addition, the role of iNOS has been examined in vivo in animal models of inflammation using pharmacological iNOS inhibitors. These experiments were extended by the advent of the iNOS-deficient (iNOS-/-) mouse. Intravital microscopy studies of these mice have indicated that, under conditions of low-dose endotoxaemia, iNOS-derived nitric oxide can inhibit leucocyte rolling and adhesion. The potential mechanisms for these effects are discussed. In contrast, several other studies have observed either no effect or an enhancing effect of iNOS on inflammatory leucocyte recruitment. Taken together, these studies suggest that the importance of iNOS in modulating leucocyte recruitment can vary according to the type of inflammatory response.

scholarly journals Genetic alteration of endothelial heparan sulfate selectively inhibits tumor angiogenesis

2007 ◽  
Vol 177 (3) ◽  
pp. 539-549 ◽  
Author(s):  
Mark M. Fuster ◽  
Lianchun Wang ◽  
Janice Castagnola ◽  
Lyudmila Sikora ◽  
Krisanavane Reddi ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Heparan Sulfate ◽  
Tumor Angiogenesis ◽  
Wound Repair ◽  
Reproductive Capacity ◽  
Erk Phosphorylation ◽  
Tumor Endothelium ◽  
A Cell

To examine the role of endothelial heparan sulfate during angiogenesis, we generated mice bearing an endothelial-targeted deletion in the biosynthetic enzyme N-acetylglucosamine N-deacetylase/N-sulfotransferase 1 (Ndst1). Physiological angiogenesis during cutaneous wound repair was unaffected, as was growth and reproductive capacity of the mice. In contrast, pathological angiogenesis in experimental tumors was altered, resulting in smaller tumors and reduced microvascular density and branching. To simulate the angiogenic environment of the tumor, endothelial cells were isolated and propagated in vitro with proangiogenic growth factors. Binding of FGF-2 and VEGF164 to cells and to purified heparan sulfate was dramatically reduced. Mutant endothelial cells also exhibited altered sprouting responses to FGF-2 and VEGF164, reduced Erk phosphorylation, and an increase in apoptosis in branching assays. Corresponding changes in growth factor binding to tumor endothelium and apoptosis were also observed in vivo. These findings demonstrate a cell-autonomous effect of heparan sulfate on endothelial cell growth in the context of tumor angiogenesis.

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