Culture of Human Endothelial Cells Derived from Umbilical Veins. IDENTIFICATION BY MORPHOLOGIC AND IMMUNOLOGIC CRITERIA

Jaffe, Eric; Nachman, Ralph L.; Becker, Carl G.; Minick, C R

doi:10.1172/jci107470

Cited by 6,471 publications

(2,752 citation statements)

References 29 publications

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“…1973). Cells were seeded onto 25 cm 2 plastic culture flasks precoated with 1% gelatine containing M199 supplemented with 20% heat‐inactivated fetal calf serum, 1 mmol l −1 sodium pyruvate, antibiotics (150 U ml −1 penicillin–150 pg ml −1 streptomycin) and 10 ng ml −1 heparin‐bound (50 μg ml −1 ) endothelial growth factor.…”

Section: Methodsmentioning

confidence: 99%

Mechanisms for the control of local tissue blood flow during thermal interventions: influence of temperature‐dependent ATP release from human blood and endothelial cells

Kalsi

Chiesa

Trangmar

et al. 2017

Experimental Physiology

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New Findings What is the central question of this study? Skin and muscle blood flow increases with heating and decreases with cooling, but the temperature‐sensitive mechanisms underlying these responses are not fully elucidated. What is the main finding and its importance? We found that local tissue hyperaemia was related to elevations in ATP release from erythrocytes. Increasing intravascular ATP augmented skin and tissue perfusion to levels equal or above thermal hyperaemia. ATP release from isolated erythrocytes was altered by heating and cooling. Our findings suggest that erythrocytes are involved in thermal regulation of blood flow via modulation of ATP release. Local tissue perfusion changes with alterations in temperature during heating and cooling, but the thermosensitivity of the vascular ATP signalling mechanisms for control of blood flow during thermal interventions remains unknown. Here, we tested the hypotheses that the release of the vasodilator mediator ATP from human erythrocytes, but not from endothelial cells or other blood constituents, is sensitive to both increases and reductions in temperature and that increasing intravascular ATP availability with ATP infusion would potentiate thermal hyperaemia in limb tissues. We first measured blood temperature, brachial artery blood flow and plasma [ATP] during passive arm heating and cooling in healthy men and found that they increased by 3.0 ± 1.2°C, 105 ± 25 ml min−1 °C−1 and twofold, respectively, (all P < 0.05) with heating, but decreased or remained unchanged with cooling. In additional men, infusion of ATP into the brachial artery increased skin and deep tissue perfusion to levels equal or above thermal hyperaemia. In isolated erythrocyte samples exposed to different temperatures, ATP release increased 1.9‐fold from 33 to 39°C (P < 0.05) and declined by ∼50% at 20°C (P < 0.05), but no changes were observed in cultured human endothelial cells, plasma or serum samples. In conclusion, increases in plasma [ATP] and skin and deep tissue perfusion with limb heating are associated with elevations in ATP release from erythrocytes, but not from endothelial cells or other blood constituents. Erythrocyte ATP release is also sensitive to temperature reductions, suggesting that erythrocytes may function as thermal sensors and ATP signalling generators for control of tissue perfusion during thermal interventions.

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Section: Methodsmentioning

confidence: 99%

Mechanisms for the control of local tissue blood flow during thermal interventions: influence of temperature‐dependent ATP release from human blood and endothelial cells

Kalsi

Chiesa

Trangmar

et al. 2017

Experimental Physiology

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“…HUVEC were isolated and cultured using a modified method described by Jaffe et al [49]. For induction studies human rIFNb-1a (Avonex s , Biogen, MA USA or Rebif s , Serono, Geneva, Switzerland) and rIFN-b-1b (Betaferon s , Schering, Berlin, Germany) were used.…”

Section: Antibodies Cells and Reagentsmentioning

confidence: 99%

“…Astrocytes were cultured as previously described [47,48] in complete DMEM media (Invitrogen, Burlington, ON, Canada) supplemented with 10% FBS (Sigma, Oakville, ON, Canada). Astrocyte-conditioned media was harvested once a week from confluent flasks and added to the BBB-EC culture media when specified.HUVEC were isolated and cultured using a modified method described by Jaffe et al [49]. For induction studies human rIFNb-1a (Avonex s , Biogen, MA USA or Rebif s , Serono, Geneva, Switzerland) and rIFN-b-1b (Betaferon s , Schering, Berlin, Germany) were used.…”

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confidence: 99%

IFN‐β regulates CD73 and adenosine expression at the blood–brain barrier

Niemelä¹,

Ifergan²,

Yegutkin³

et al. 2008

Eur J Immunol

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IFN-b treatment reduces the relapse rate in MS but its mechanism of action remains incompletely understood. Our aim was to clarify the beneficial effect of IFN-b in the treatment of MS. We assessed the influence of IFN-b treatment on (i) CD73 expression on the surface of primary cultures of human blood-brain barrier endothelial cells (BBB-EC) and human astrocytes using immunofluorescence staining and flow cytometry, (ii) transmigration of CD4 1 T lymphocytes using an in vitro model of BBB and (iii) CD73 enzyme activity, i.e. ecto-5 0 -nucleotidase activity in the serum of MS patients using a radiochemical assay. IFN-b increases the expression of ecto-5 0 -nucleotidase both on BBB-EC and astrocytes. As a consequence, lymphocyte transmigration through BBB-EC is reduced. Importantly, this reduction can be reversed using a,b-methyleneadenosine-5 0 -diphosphate, a specific inhibitor of ecto-5 0 -nucleotidase. CD73 is strongly expressed in microvasculature in samples of postmortem MS brain and, moreover, in the majority of MS patients there was a clear upregulation both in the soluble serum ecto-5 0 -nucleotidase activity and skin microvascular CD73 expression after IFN-b treatment. Upregulation of ecto-5 0 -nucleotidase and a subsequent increase in adenosine production might contribute to the beneficial effects of IFN-b on MS via enhancing the endothelial barrier function.Key words: Adenosine . Blood-brain barrier . CD73 . IFN-b . MS Introduction MS is a demyelinating disease characterized by perivascular inflammatory cell infiltrates in the white matter of the CNS. MS is generally thought to be an autoimmune disease in which the immune system attack against self-antigens results in inflammation and neurological symptoms [1]. First line treatment of MS is subcutaneous or intramuscular administration of IFN-b, which leads to a reduced number of relapses and a slower disease progression [2][3][4]. The proposed beneficial effects of IFN-b include a decrease in antigen presentation, MHC class II expression, T-cell proliferation, IFN-b production, ECM degradation, and the expression of adhesion molecules [5]. Moreover, recent reports describe IFN-b as a suppressor of both myeloid [6] and Th17-cells [7]. IFN-b also reduces the number of gadolinium enhancing lesions in brain magnetic resonance imaging of MS patients [2,3,8], which is thought to reflect its ability to regulate the blood-brain barrier (BBB) function [9]. Ecto-5 0 -nucleotidase (CD73; EC 3.1.3.5) is a 70 kDa GPI-anchored glycoprotein. It is abundantly expressed on vascular endothelium and subpopulations of lymphocytes, but not on granulocytes or monocytes [10] and it also circulates in blood in a soluble 2718form [11]. Ecto-5 0 -nucleotidase activity is found on many CNS cell types such as astrocytes, oligodendrocytes, microglial cells and brain EC in many species including human, rat, mouse and bovine [12][13][14][15]. CD73 modulates the extent of immune and inflammatory responses in vitro and in vivo. These effects are mediated via its enzymatic capability ...

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“…Human umbilical vein endothelial cells (HUVECs) were isolated from cords taken from normal term deliveries by standard protocols 18 .…”

Section: Methodsmentioning

confidence: 99%

Myometrial microvascular endothelial cells express oxytocin receptor

Weston¹

2003

BJOG: An International Journal of Obstetrics and Gynaecology

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Objective 1. To establish whether microvascular endothelial cells from the human myometrium (MMECs) express oxytocin receptor, and to compare its expression levels relative to HUVECs. 2. To verify an upregulation of oxytocin receptor expression in MMECs as a result of vascular endothelial growth factor (VEGF), which had been found in a previous study. Design Laboratory scientific study. Setting University department.Population or Sample Myometrial biopsies from 12 hysterectomy specimens.Methods MMECs and HUVECs were established in vitro. Immunohistochemistry of in vitro cultures was performed to investigate protein expression of the oxytocin receptor. Semi-quantitative RT-PCR and Northern blots were performed to examine the presence and relative abundance of oxytocin receptor mRNA in MMECs and HUVECs, and in both cell types with and without VEGF. Total RNA from oxytocin acetate (100 nmol/L) and vehicle stimulated endothelial cell cultures was used to examine gene expression differences on a 10.5K cDNA microarray. Main outcome measures Oxytocin receptor mRNA and protein; gene expression analysis.Results Oxytocin receptor mRNA and protein was present in MMECs. The level of expression was the same as for the HUVECs, but much less than the pregnant myometrium. No effect on gene expression could be demonstrated by gene expression microarray following 10 hours of oxytocin stimulation. Twenty-four hours of VEGF stimulation did not significantly alter oxytocin receptor mRNA expression in MMECs or HUVECs. Conclusion The myometrial microvasculature expresses oxytocin receptor. This finding means that oxytocin may exert some of its effects on the myometrial microvasculature. No evidence could be found for a transcriptional effect of oxytocin stimulation in this study, but further work on the role of the myometrial vessel oxytocin receptor is needed.

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Culture of Human Endothelial Cells Derived from Umbilical Veins. IDENTIFICATION BY MORPHOLOGIC AND IMMUNOLOGIC CRITERIA

Abstract: A B S T R A C T Endothelial cells

Cited by 6,471 publications

References 29 publications

Mechanisms for the control of local tissue blood flow during thermal interventions: influence of temperature‐dependent ATP release from human blood and endothelial cells

Mechanisms for the control of local tissue blood flow during thermal interventions: influence of temperature‐dependent ATP release from human blood and endothelial cells

IFN‐β regulates CD73 and adenosine expression at the blood–brain barrier

Myometrial microvascular endothelial cells express oxytocin receptor

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