HUMAN ENDOTHELIAL CELLS MODIFICATIONS DURING LONG TERM CULTURE

1987 ◽  
Author(s):  
M P Wautier ◽  
J L Wautier
Keyword(s):  
Endothelial Cells ◽  
Doubling Time ◽  
Umbilical Vein ◽  
Light And Electron Microscopy ◽  
Major Change ◽  
Culture Conditions ◽  
Human Endothelial Cells ◽  
Long Term Culture

The culture of human endothelial cells is largely used for vascular research. The possibility of developping long term culture of human endothelial cells (EC) raised the question regarding the identity after several passages. To further investigate this aspect we have cultured human umbilical vein EC until the 12th passage on fibronectin coated dishes supplemented with ECGF. We have studied the EC morphology by light and electron microscopy, the reactivity with 51Cr labelled platelets, and prostacyclin synthesis. Until the 6th passage no major change could be noted, except the occurence of rare large EC and a reduction in the doubling time between 2nd and 5th passage. After the 7th passage up to the 10th EC became more elongated and did not grow in strict monolayer. The number of vacuoles and mitochondria increased as well as the doubling time. After the 12th passage the EC were still viable but proliferated very slowly. The adhesion of radiolabelled platelets dramatically increased (150%) and PGI2 production significantly decreased (6 Keto PGF1α : 1st passage 13±2.5 ng; 6th passage 0.33±0.27 ng/106 EC). In our culture conditions EC kept most of their original characteristics up to the 6th passage but then lost some of them. At any passage EC contained Weibel Palade bodies and von Willebrand factor. We can conclude that after the 7th passage EC in culture are different from the original cells and could possibly represent an in vitro model of EC ageing.

Cells derived from porcine aorta tunica media show mesenchymal stromal-like cell properties in in vitro culture

2014 ◽  
Vol 306 (4) ◽  
pp. C322-C333 ◽  
Author(s):  
Andrea Zaniboni ◽  
Chiara Bernardini ◽  
Marco Alessandri ◽  
Chiara Mangano ◽  
Augusta Zannoni ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
In Vitro Culture ◽  
Umbilical Vein ◽  
Culture Conditions ◽  
Differentiation Potential ◽  
Tunica Media ◽  
Large Numbers ◽  
Umbilical Vein Endothelial Cells ◽  
Pig Animal Model

Several studies have already described the presence of specialized niches of precursor cells in vasculature wall, and it has been shown that these populations share several features with mesenchymal stromal cells (MSCs). Considering the relevance of MSCs in the cardiovascular physiopathology and regenerative medicine, and the usefulness of the pig animal model in this field, we reported a new method for MSC-like cell isolation from pig aorta. Filling the vessel with a collagenase solution for 40 min, all endothelial cells were detached and discarded and then collagenase treatment was repeated for 4 h to digest approximately one-third of the tunica media. The ability of our method to select a population of MSC-like cells from tunica media could be ascribed in part to the elimination of contaminant cells from the intimal layer and in part to the overnight culture in the high antibiotic/antimycotic condition and to the starvation step. Aortic-derived cells show an elongated, spindle shape, fibroblast-like morphology, as reported for MSCs, stain positively for CD44, CD56, CD90, and CD105; stain negatively for CD34 and CD45; and express CD73 mRNA. Moreover, these cells show the classical mesenchymal trilineage differentiation potential. Under our in vitro culture conditions, aortic-derived cells share some phenotypical features with pericytes and are able to take part in the formation of network-like structures if cocultured with human umbilical vein endothelial cells. In conclusion, our work reports a simple and highly suitable method for obtaining large numbers of precursor MSC-like cells derived from the porcine aortic wall.

scholarly journals AICAR Protects Vascular Endothelial Cells from Oxidative Injury Induced by the Long-Term Palmitate Excess

2021 ◽  
Vol 23 (1) ◽  
pp. 211
Author(s):  
Mikhail V. Samsonov ◽  
Nikita V. Podkuychenko ◽  
Asker Y. Khapchaev ◽  
Eugene E. Efremov ◽  
Elena V. Yanushevskaya ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Insulin Signaling ◽  
Umbilical Vein ◽  
Blood Levels ◽  
Ros Generation ◽  
No Production ◽  
Vascular Endothelial ◽  
Long Term Effects

Hyperlipidemia manifested by high blood levels of free fatty acids (FFA) and lipoprotein triglycerides is critical for the progression of type 2 diabetes (T2D) and its cardiovascular complications via vascular endothelial dysfunction. However, attempts to assess high FFA effects in endothelial culture often result in early cell apoptosis that poorly recapitulates a much slower pace of vascular deterioration in vivo and does not provide for the longer-term studies of endothelial lipotoxicity in vitro. Here, we report that palmitate (PA), a typical FFA, does not impair, by itself, endothelial barrier and insulin signaling in human umbilical vein endothelial cells (HUVEC), but increases NO release, reactive oxygen species (ROS) generation, and protein labeling by malondialdehyde (MDA) hallmarking oxidative stress and increased lipid peroxidation. This PA-induced stress eventually resulted in the loss of cell viability coincident with loss of insulin signaling. Supplementation with 5-aminoimidazole-4-carboxamide-riboside (AICAR) increased endothelial AMP-activated protein kinase (AMPK) activity, supported insulin signaling, and prevented the PA-induced increases in NO, ROS, and MDA, thus allowing to maintain HUVEC viability and barrier, and providing the means to study the long-term effects of high FFA levels in endothelial cultures. An upgraded cell-based model reproduces FFA-induced insulin resistance by demonstrating decreased NO production by vascular endothelium.

scholarly journals Infection of Human Endothelial Cells with Chlamydia pneumoniae Stimulates Transendothelial Migration of Neutrophils and Monocytes

1999 ◽  
Vol 67 (3) ◽  
pp. 1323-1330 ◽  
Author(s):  
Robert E. Molestina ◽  
Richard D. Miller ◽  
Julio A. Ramirez ◽  
James T. Summersgill
Keyword(s):  
Endothelial Cells ◽  
Chlamydia Pneumoniae ◽  
Umbilical Vein ◽  
Neutrophil Migration ◽  
Transendothelial Migration ◽  
Serial Passage ◽  
Human Endothelial Cells ◽  
Monocyte Migration ◽  
Low Passage

ABSTRACT We have previously shown that different isolates of Chlamydia pneumoniae display heterogeneity in the in vitro stimulation of chemokines and adhesion molecules from infected human endothelial cells. In the present study, we examined the ability of different isolates of C. pneumoniae to promote transendothelial migration of neutrophils and monocytes. Human umbilical vein endothelial cells (HUVEC) were infected with low (<15)-passageC. pneumoniae isolates A-03, PS-32, and BR-393 and high (>40)-passage isolates BAL-16, TW-183, and T-2634, and levels of neutrophil and monocyte transendothelial migration were determined following 24 h of infection. Compared to mock-infected controls, significant increases in neutrophil migration were observed in response to most C. pneumoniae isolates examined (P < 0.001). Levels of monocyte migration were significantly increased in response to TW-183 and T-2634 (P < 0.001). Serial passage (>40 times) of the three low-passage isolates in HEp-2 cell cultures prior to infection of HUVEC generally resulted in the promotion of higher levels of neutrophil and monocyte transendothelial migration. These findings were compatible with differences observed in the extent of interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) stimulation between low- and high-passage A-03, PS-32, and BR-393. As opposed toC. pneumoniae, infection with C. trachomatis L2 caused only a slight increase in neutrophil transendothelial migration, which correlated with the lack of measurable IL-8 levels by this species. However, significant levels of monocyte migration were induced in response to C. trachomatis L2 despite a lack of measurable MCP-1 stimulation.C. trachomatis serovars A and E also failed to induce IL-8 and MCP-1 production in HUVEC. Results from this study indicate that the passage history of C. pneumoniae may play a role in the divergence of stimulatory activities observed among isolates in human endothelial cells. In addition, the differences observed between this organism and C. trachomatissuggest that the upregulation of IL-8 and MCP-1 in endothelial cells may be unique to C. pneumoniae.

A proteasome inhibitor reduces concurrent, sequential, and long-term IL-1β- and TNF-α-induced ECAM expression and adhesion

2003 ◽  
Vol 285 (4) ◽  
pp. C813-C822 ◽  
Author(s):  
Nilesh M. Dagia ◽  
Douglas J. Goetz
Keyword(s):  
Endothelial Cells ◽  
Cell Adhesion ◽  
Proteasome Inhibitor ◽  
Umbilical Vein ◽  
Hl60 Cell ◽  
Induced Expression ◽  
Tnf Α ◽  
Short Period

A promising approach for reducing aberrant leukocyte-endothelial adhesion during pathological inflammation is to inhibit endothelial cell adhesion molecule (ECAM) expression at the transcription level. Several compounds have been shown to decrease cytokine-induced upregulation of ECAMs primarily by modulating the activity of transcription factors [e.g., nuclear factor-κB (NF-κB)]. The majority of the in vitro studies have focused on the effect of transcription inhibitors on endothelial cells exposed to a single cytokine [primarily tumor necrosis factor-α (TNF-α)] for a relatively short period of time (primarily 4-6 h). However, in the in vivo setting, multiple cytokines [e.g., interleukin-1β (IL-1β) and TNF-α] may be present for extended periods of time. Thus we studied the effects of a transcription inhibitor, the proteasome inhibitor lactacystin, on ECAM expression and myeloid (HL60) cell adhesion to human umbilical vein endothelial cells (HUVEC) activated by concurrent, sequential, and long-term (24 h) treatment with IL-1β and TNF-α. We show, for the first time, that lactacystin inhibits 1) 4-h concurrent IL-1β- and TNF-α-induced expression of E-selectin, VCAM-1, ICAM-1, and HL60 cell adhesion to HUVEC; 2) 4-h TNF-α-induced expression of E-selectin, VCAM-1, and HL60 cell adhesion to HUVEC that have become desensitized to IL-1β activation; 3) 24-h TNF-α-induced expression of E-selectin and VCAM-1 but not ICAM-1; and 4) 24-h TNF-α-induced HL60 cell adhesion to HUVEC. Combined, our results demonstrate that a proteasome inhibitor can reduce concurrent, sequential, and long-term IL-1β- and TNF-α-induced ECAM expression and myeloid cell adhesion.

scholarly journals CD44 Is Involved in Tumor Angiogenesis; an Activation Antigen on Human Endothelial Cells

Blood ◽  
1997 ◽  
Vol 90 (3) ◽  
pp. 1150-1159 ◽  
Author(s):  
Arjan W. Griffioen ◽  
Marieke J.H. Coenen ◽  
Cora A. Damen ◽  
Sandra M.M. Hellwig ◽  
David H.J. van Weering ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Growth Factor ◽  
Tumor Angiogenesis ◽  
Umbilical Vein ◽  
Human Endothelial Cells ◽  
Specific Expression ◽  
Cd44 Expression ◽  
Activation Antigen

Abstract CD44 is described to be an activation molecule in a number of different cell types. We investigated the role of CD44 on human endothelial cells (EC) and in tumor angiogenesis. Using flow cytometry we showed that EC from the vasculature of human solid tumors display an enhanced expression of CD44 as compared to EC from normal tissue. This finding was confirmed by immunohistochemical studies on frozen tissue sections. Because tumors are dependent on angiogenesis, the role of angiogenic stimuli in the enhanced CD44 expression was investigated. We found that basic fibroblast growth factor (bFGF ) and vascular endothelial growth factor were able to efficiently upregulate CD44 expression on cultured human EC. The upregulation reached maximal levels after treatment for 3 days with 10 ng/mL bFGF. The physiological impact of this upregulation was shown by the enhanced binding of EC to hyaluronate after pretreatment with bFGF. In a next set of studies that were designed to unravel the regulation of CD44 expression on EC we concluded that CD44 is an activation antigen on human EC since (1) human umbilical vein derived endothelial cells, which in vivo do not express CD44, begin to express CD44 when plated and cultured, (2) CD44 expression is enhanced after subculture of confluent cultures, (3) CD44 is predominantly expressed on the BrdU incorporating subset of cultured EC. The specific expression of CD44 on activated and tumor EC prompted us to study the usefulness of CD44 as an endothelial target for therapy with immunotoxins. In vitro experiments showed that EC are efficiently killed after targeting immunotoxin to CD44.

scholarly journals Poloxamer 407-induced atherosclerosis in mice appears to be due to lipid derangements and not due to its direct effects on endothelial cells and macrophages

2003 ◽  
Vol 12 (3) ◽  
pp. 147-155 ◽  
Author(s):  
Thomas P. Johnston ◽  
Yuai Li ◽  
Ahmed S. Jamal ◽  
Daniel J. Stechschulte ◽  
Kottarappat N. Dileepan
Keyword(s):  
Endothelial Cells ◽  
Mouse Model ◽  
Umbilical Vein ◽  
Density Lipoprotein ◽  
Poloxamer 407 ◽  
Direct Effects ◽  
Direct Stimulation

Coronary heart disease secondary to atherosclerosis is still the leading cause of death in the US. Animal models used for elucidating the pathogenesis of this disease primarily involve rabbits and pigs. Previous studies from this laboratory have demonstrated intraperitoneal injections of poloxamer 407 (P-407) in both male and female mice will lead to hyperlipidemia and atherosclerosis, suggesting the use of this polymer to develop a mouse model of atherosclerosis. In order to understand the mechanism of P-407-induced hyperlipidemia and vascular lesion formation, we evaluated the direct effects of P-407 on endothelial cell and macrophage functionsin vitro, and itsin vivoeffects on the oxidation of circulating lipids following long-term (4 month) administration. Our results demonstrated that incubation of P-407 with human umbilical vein endothelial cells in culture did not influence either cell proliferation or interleukin-6 and interleukin-8 production over a concentration range of 0-40 μM. In addition, nitric oxide production by macrophages was not affected by P-407 over a concentration range of 0-20 μM. Finally, we demonstrated that while P-407 could not induce the oxidation of LDL-Cin vitro, long-term (4 month) administration of P-407 in mice resulted in elevated levels of oxidized lipids in the plasma. Thus, it is suggested that the formation of atherosclerotic lesions in this mouse model of atherosclerosis does not result from either direct stimulation of endothelial cells or macrophage activation by P-407. Instead, these data would support the premise that oxidation of lipids (perhaps low-density lipoprotein cholesterol) by an indirect mechanism following injection of P-407 may represent one of the mechanisms responsible for atheroma formation.

scholarly journals Increased susceptibility of human endothelial cells to infections by SARS-CoV-2 variants

2021 ◽  
Vol 116 (1) ◽  
Author(s):  
Julian U. G. Wagner ◽  
Denisa Bojkova ◽  
Mariana Shumliakivska ◽  
Guillermo Luxán ◽  
Luka Nicin ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Dysfunction ◽  
Umbilical Vein ◽  
Cell Number ◽  
Spike Protein ◽  
Human Endothelial Cells ◽  
Health Crisis ◽  
Novel Coronavirus ◽  
Umbilical Vein Endothelial Cells

AbstractCoronavirus disease 2019 (COVID-19) spawned a global health crisis in late 2019 and is caused by the novel coronavirus SARS-CoV-2. SARS-CoV-2 infection can lead to elevated markers of endothelial dysfunction associated with higher risk of mortality. It is unclear whether endothelial dysfunction is caused by direct infection of endothelial cells or is mainly secondary to inflammation. Here, we investigate whether different types of endothelial cells are susceptible to SARS-CoV-2. Human endothelial cells from different vascular beds including umbilical vein endothelial cells, coronary artery endothelial cells (HCAEC), cardiac and lung microvascular endothelial cells, or pulmonary arterial cells were inoculated in vitro with SARS-CoV-2. Viral spike protein was only detected in HCAECs after SARS-CoV-2 infection but not in the other endothelial cells tested. Consistently, only HCAEC expressed the SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2), required for virus infection. Infection with the SARS-CoV-2 variants B.1.1.7, B.1.351, and P.2 resulted in significantly higher levels of viral spike protein. Despite this, no intracellular double-stranded viral RNA was detected and the supernatant did not contain infectious virus. Analysis of the cellular distribution of the spike protein revealed that it co-localized with endosomal calnexin. SARS-CoV-2 infection did induce the ER stress gene EDEM1, which is responsible for clearance of misfolded proteins from the ER. Whereas the wild type of SARS-CoV-2 did not induce cytotoxic or pro-inflammatory effects, the variant B.1.1.7 reduced the HCAEC cell number. Of the different tested endothelial cells, HCAECs showed highest viral uptake but did not promote virus replication. Effects on cell number were only observed after infection with the variant B.1.1.7, suggesting that endothelial protection may be particularly important in patients infected with this variant.

scholarly journals C-Type Natriuretic Peptide (CNP) Inhibition of Interferon-γ-Mediated Gene Expression in Human Endothelial Cells In Vitro

Biosensors ◽  
2018 ◽  
Vol 8 (3) ◽  
pp. 86
Author(s):  
Amy Day ◽  
Zoe Jameson ◽  
Carolyn Hyde ◽  
Bigboy Simbi ◽  
Robert Fowkes ◽  
...  
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Inflammatory Responses ◽  
Umbilical Vein ◽  
Vascular Inflammation ◽  
Western World ◽  
Human Endothelial Cells ◽  
Ifn Γ

Cardiovascular diseases, including atherosclerosis, now account for more deaths in the Western world than from any other cause. Atherosclerosis has a chronic inflammatory component involving Th1 pro-inflammatory cytokines such as IFN-γ, which is known to induce endothelial cell inflammatory responses. On the other hand CNP, which acts via its receptors to elevate intracellular cGMP, is produced by endothelium and endocardium and is upregulated in atherosclerosis. It is believed to be protective, however its role in vascular inflammation is not well understood. The aim of this study was to investigate the effects of CNP on human endothelial cell inflammatory responses following IFN-γ stimulation. Human umbilical vein endothelial cells were treated with either IFN-γ (10 ng/mL) or CNP (100 nm), or both in combination, followed by analysis by flow cytometry for expression of MHC class I and ICAM-1. IFN-γ significantly increased expression of both molecules, which was significantly inhibited by CNP or the cGMP donor 8-Bromoguanosine 3’,5’-cyclic monophosphate (1 µm). CNP also reduced IFN-γ mediated kynurenine generation by the IFN-γ regulated enzyme indoleamine-2,3-deoxygenase (IDO). We conclude that CNP downmodulates IFN-γ induced pro-inflammatory gene expression in human endothelial cells via a cGMP-mediated pathway. Thus, CNP may have a protective role in vascular inflammation and novel therapeutic strategies for CVD based on upregulation of endothelial CNP expression could reduce chronic EC inflammation.

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.

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