Vascular endothelium as a regulator of granulopoiesis: production of colony-stimulating activity by cultured human endothelial cells

Colony-stimulating activity is a regulatory factor(s) that promotes differentiation of hemopoietic stem cells to mature granulocytes and macrophages; in man it has been found that blood monocytes, lymphocytes, and tissue macrophages produce it. In an effort to identify other potenitally physiologic tissue sources of colony- stimulating activity, we have studied the capacity of primary cultures of human vascular endothelial cells to produce colony-stimulating activity. Medium conditioned by incubation with endothelial cultures contained activity that promoted granulocyte-macrophage colony formation of nonadherent human and murine marrow cells. Exposure of endothelial cultures to 0.1–5.0 microgram/ml S. typhosa endotoxin for 6- 72 hr enhanced colony-stimulating activity production. Similarly, incubation of endothelial cells with lysates of human blood granulocytes, or cocultivation with intact granulocytes, resulted in increased colony-stimulating activity levels. In 7–14 day cultures, freshly isolated endothelial cells, incorporated into agar underlayers, consistently stimulated more colony formation by nonadherent human marrow cells than comparable numbers of blood monocytes. These data indicate that: (1) cultured human endothelial cells are a potent source of colony-stimulating activity; (2) they respond to endotoxin and granulocytes and their contents by producing increased amounts of CSA; and (3) they produce morea colony-stimulating activity, than human blood monocytes under standardized conditions in vitro. These observations suggest that the vascular endothelium may play a role in the physiologic regulation of granulopoiesis.

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Vascular endothelium as a regulator of granulopoiesis: production of colony-stimulating activity by cultured human endothelial cells

Blood ◽

10.1182/blood.v56.6.1060.1060 ◽

1980 ◽

Vol 56 (6) ◽

pp. 1060-1067 ◽

Cited By ~ 75

Author(s):

PJ Quesenberry ◽

MA Jr Gimbrone

Keyword(s):

Endothelial Cells ◽

Human Blood ◽

Vascular Endothelium ◽

Colony Formation ◽

Primary Cultures ◽

Blood Monocytes ◽

Human Endothelial Cells ◽

Colony Stimulating Activity ◽

Marrow Cells

Abstract Colony-stimulating activity is a regulatory factor(s) that promotes differentiation of hemopoietic stem cells to mature granulocytes and macrophages; in man it has been found that blood monocytes, lymphocytes, and tissue macrophages produce it. In an effort to identify other potenitally physiologic tissue sources of colony- stimulating activity, we have studied the capacity of primary cultures of human vascular endothelial cells to produce colony-stimulating activity. Medium conditioned by incubation with endothelial cultures contained activity that promoted granulocyte-macrophage colony formation of nonadherent human and murine marrow cells. Exposure of endothelial cultures to 0.1–5.0 microgram/ml S. typhosa endotoxin for 6- 72 hr enhanced colony-stimulating activity production. Similarly, incubation of endothelial cells with lysates of human blood granulocytes, or cocultivation with intact granulocytes, resulted in increased colony-stimulating activity levels. In 7–14 day cultures, freshly isolated endothelial cells, incorporated into agar underlayers, consistently stimulated more colony formation by nonadherent human marrow cells than comparable numbers of blood monocytes. These data indicate that: (1) cultured human endothelial cells are a potent source of colony-stimulating activity; (2) they respond to endotoxin and granulocytes and their contents by producing increased amounts of CSA; and (3) they produce morea colony-stimulating activity, than human blood monocytes under standardized conditions in vitro. These observations suggest that the vascular endothelium may play a role in the physiologic regulation of granulopoiesis.

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

10.1101/2020.12.02.391664 ◽

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.

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Human Blood Monocytes: Stimulators of Granulocyte and Mononuclear Colony Formation in vitro

Science ◽

10.1126/science.178.4057.164 ◽

1972 ◽

Vol 178 (4057) ◽

pp. 164-166 ◽

Cited By ~ 236

Author(s):

P. A. Chervenick ◽

A. F. LoBuglio

Keyword(s):

Human Blood ◽

Colony Formation ◽

Blood Monocytes

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Specificity of the Thrombin-Induced Release of Tissue Plasminogen Activator from Cultured Human Endothelial Cells

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661622 ◽

1986 ◽

Vol 56 (02) ◽

pp. 115-119 ◽

Cited By ~ 43

Author(s):

Eugene G Levin ◽

David M Stern ◽

Peter P Nawroth ◽

Richard A Marlar ◽

Daryl S Fair ◽

...

Keyword(s):

Endothelial Cells ◽

Plasminogen Activator ◽

Tissue Plasminogen Activator ◽

Protein C ◽

Primary Cultures ◽

Activated Protein C ◽

Specific Inhibitor ◽

Factor Xa ◽

Human Endothelial Cells ◽

Tissue Plasminogen

SummaryThe addition of thrombin (9 nM) to primary cultures of human endothelial cells induces a 6- to 7-fold increase in the rate of release of tissue plasminogen activator (tPA). Several other serine proteases which specifically interact with endothelial cells were also analyzed for their effect on tPA release. Gamma-thrombin, an autocatalytic product of α-thrombin, promoted tPA release but was less effective than α-thrombin. A maximum increase of 5.5-fold was observed, although a concentration of γ-thrombin 20 times greater than α-thrombin was required. The response to Factor Xa was similar to α-thrombin, although the stimulation was significantly reduced by the addition of hirudin or DAPA suggesting that prothrombin activation was occurring. The simultaneous addition of prothrombin with Factor Xa resulted in enhanced tPA release equal to that observed with an equimolar concentration of active α-thrombin. Thus, under these conditions, Factor Xa-cell surface mediated activation of prothrombin can lead to a secondary effect resulting from cell-thrombin interaction. Activated protein C, which has been implicated as a profibrinolytic agent, was also tested. No change in tPA release occurred after the addition of up to 325 nM activated protein C in the presence or absence of proteins. Factor IXa and plasmin were also ineffective. The effect of thrombin on the endothelial cell derived plasminogen activator specific inhibitor was also studied. Thrombin produced a small but variable release of the inhibitor with an increase of less than twice that of non-thrombin treated controls.

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Interactions Between Blood Platelets and Vascular Endothelium in vitro Studies

10.1055/s-0039-1684685 ◽

1979 ◽

Author(s):

M.A. Gimbrone ◽

K.D. Curwen ◽

R. I. Handin

Keyword(s):

Platelet Aggregation ◽

Vascular Endothelium ◽

Potent Inhibitor ◽

Platelet Reactivity ◽

Blood Platelets ◽

Primary Cultures ◽

Adenosine Diphosphate ◽

Human Platelets ◽

Platelet Adherence

Endothelial cells (EC) can actively influence the hemostatic response at sites of vascular injury through multiple mechanisms. For example, EC can degrade adenosine diphosphate, release plasminogen activator, and synthesize prostacyclin (PGI2), a potent inhibitor of platelet aggregation. We have examined whether PGI2 also might account for the normal lack of platelet adherence to the uninjured EC surface. In a monolayer adherence assay, radiolabeled human platelets in citrated plasma showed minimal interaction with primary cultures of human EC (<1 platelet adhering per cell). Platelets from aspirin-treated and untreated donors behaved similarly. However, aspirin pretreatment of EC consistently resulted in ~2-fold increases in platelet adherence which could be completely abolished by exogenous PGI2 (0.5–1.0 μg/ml). SV40-transformed human EC (SVHEC), which are deficient in PGI2 production compared to primary EC, showed 10-30 times more platelet adherence. Exogenous PGI2 produced a dose - related (.001-1.0 μg/ml) decrease in platelet adherence to SVHEC but did not result in the basal levels observed with normal EC monolayers. These data suggest that : 1) In addition to its effects on platelet aggregation, PGI2 can influence platelet endothelial cell interactions; 2) The increased platelet reactivity of transformed EC is associated with, but not completely attributable, to decreased PGI2 production; and 3) Factors other than PGI2 may play a role in the thromboresistance of normal vascular endothelium.

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Acute damage to human endothelial cells by brief exposure to contrast media in vitro.

Radiology ◽

10.1148/radiology.147.3.6844604 ◽

1983 ◽

Vol 147 (3) ◽

pp. 681-684 ◽

Cited By ~ 50

Author(s):

F Laerum

Keyword(s):

Endothelial Cells ◽

Contrast Media ◽

Human Endothelial Cells

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Vascular repair utilising immobilised heparin conjugate for protection against early activation of inflammation and coagulation

Thrombosis and Haemostasis ◽

10.1160/th14-09-0724 ◽

2015 ◽

Vol 113 (06) ◽

pp. 1312-1322 ◽

Cited By ~ 18

Author(s):

Sofia Nordling ◽

Jaan Hong ◽

Karin Fromell ◽

Fredrik Edin ◽

Johan Brännström ◽

...

Keyword(s):

Endothelial Cells ◽

Thrombus Formation ◽

Innate Immune ◽

Human Endothelial Cells ◽

Coagulation Activation ◽

Ischaemia Reperfusion Injury ◽

Novel Approach ◽

One Step ◽

Chamber Model

SummaryIschaemia-reperfusion injury (IRI) poses a major challenge in many thrombotic conditions and in whole organ transplantation. Activation of the endothelial cells and shedding of the protective vascular glycocalyx during IRI increase the risk of innate immune activation, cell infiltration and severe thrombus formation, promoting damage to the tissue. Here, we present a novel one-step strategy to protect the vasculature by immobilisation of a unique multi-arm heparin conjugate to the endothelium. Applying a new in vitro blood endothelial cell chamber model, the heparin conjugate was found to bind not only to primary human endothelial cells but also directly to the collagen to which the cells adhered. Incubation of hypoxic endothelial cells with freshly drawn human blood in the blood chambers elicited coagulation activation reflected by thrombin anti-thrombin formation and binding of platelets and neutrophils. Immobilisation of the heparin conjugate to the hypoxic endothelial cells created a protective coating, leading to a significant reduction of the recruitment of blood cells and coagulation activation compared to untreated hypoxic endothelial cells. This novel approach of immobilising multi-arm heparin conjugates on the endothelial cells and collagen of the basement membrane ensures to protect the endothelium against IRI in thrombotic disorders and in transplantation.

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Cycle initiation and colony formation in culture by murine marrow cells with long-term reconstituting potential in vivo

Blood ◽

10.1182/blood.v88.11.4149.4149 ◽

1996 ◽

Vol 88 (11) ◽

pp. 4149-4158 ◽

Cited By ~ 3

Author(s):

M Trevisan ◽

XQ Yan ◽

NN Iscove

Keyword(s):

Colony Formation ◽

Interleukin 1 ◽

Methyl Cellulose ◽

Culture Conditions ◽

Liquid Suspension ◽

Almost All ◽

Marrow Cells

Abstract This investigation was directed at separating long-term reconstituting (LTR) stem cells in normal murine marrow from hematopoietic precursors detectable in short-term assays in vitro and in vivo, and then at determining whether purified LTR cells could themselves form colonies in culture. To do so, it was first necessary to identify culture conditions that would induce their growth while preserving their long- term reconstituting capacity. Marrow was cultured with various cytokines in liquid suspension for 4 days, after which the surviving LTR activity was quantitated in a competitive in vivo assay. Activity was preserved near input levels with combined murine c-kit ligand (KL), interleukin-1 (IL-1), IL-6, and IL-11. When the cultures also included tritiated or unlabeled thymidine, LTR potential was eliminated, indicating that essentially all LTR cells were induced into cell cycle with these cytokines. To purify them, marrow was sorted on the basis of Ly6A expression and Rhodamine 123 retention. The Ly6AhiRh123ls fraction contained 85% of total recovered LTR activity but only 1% of the recovered cells measured by multilineage colony formation in spleens or in vitro. This fraction was cultured in methyl cellulose with KL, IL-1, IL-6, and IL-11 for 4 to 6 days, after which colonies were isolated and injected into mice. High levels of permanent reconstitution were achievable in sublethally irradiated W41/W41 mice after the injection of a single reconstituting unit, and limiting dilution analysis estimated the frequency of multilineage LTR at 1 in 11,200 unpurified adult marrow cells. In either lethally irradiated normal or sublethally irradiated W41/W41 mice, 1-year lymphomyeloid reconstitutions were obtained from 1 in 65 to 84 colonies of 2 to 16 dispersed cells, but not from larger colonies or those with clumped cells. The results establish that resting marrow LTR cells can be separated from almost all of the more advanced clonogenic cells that are still pluripotential, can be induced to cycle in culture by defined cytokines with preservation of their reconstituting potential, and can be manipulated and assayed efficiently at single-cell and colony levels.

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Radiocontrast agents induce endothelin release in vivo and in vitro.

Journal of the American Society of Nephrology ◽

10.1681/asn.v3158 ◽

1992 ◽

Vol 3 (1) ◽

pp. 58-65 ◽

Cited By ~ 1

Author(s):

S N Heyman ◽

B A Clark ◽

N Kaiser ◽

K Spokes ◽

S Rosen ◽

...

Keyword(s):

Endothelial Cells ◽

Plasma Concentration ◽

Plasma Level ◽

Vascular Endothelium ◽

Radiocontrast Agent ◽

Transient Rise ◽

Bovine Endothelial Cells ◽

Radiocontrast Agents

The intravascular administration of the ionic radiocontrast agent sodium iothalamate (2.9 g of iodine/kg body wt) to rats induced an increase in plasma concentration of immunoreactive endothelin from 21.3 +/- 1.2 to 36 +/- 3 fmol/mL, preceded by a transient rise in the plasma level of atrial natriuretic peptide and associated with a fall in RBF. Equi-iodine amounts of the nonionic agents ioxaglate and iohexol elicited similar or more marked changes in plasma endothelin, but hypertonic solutions of NaCl, mannitol, or glucose did not. Comparable levels of endothelin produced by infusions of endothelin-1 induced a reduction of up to 29% in RBF. Iothalamate and iohexol stimulated endothelin release from cultured bovine endothelial cells, suggesting a direct effect of ionic and nonionic agents on vascular endothelium. The data invite speculation that under some circumstances endothelin release might play a role in the circulatory changes caused by these compounds and in the pathogenesis of radiocontrast nephropathy.

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