Culture Supernatant Of Endothelial Cells Research Articles

344.5: Reduced Epidermal Growth Factor (EGF) Expression During Antibody Mediated Rejection Following Heart Transplantation

Antibody mediated rejection (AMR) remains one of the major challenges for long-term survival following heart transplantation. The immediate and long-term impact of HLA antibodies on graft survival is well established. However, the signaling pathways that are involved in AMR are not well established. Endothelial cell activation by HLA antibodies lead to the activation of several downstream signaling pathways. We utilized an in vitro primary human endothelial cell model treated with humanized monoclonal antibodies to HLA Class I antigens to identify the signaling pathways involved in AMR. A reduction of epidermal growth factor (EGF) levels in endothelial cells was observed following coupling these cells with HLA antibodies. We confirmed this result with ELISA assays of the cell culture supernatant of endothelial cells treated with humanized monoclonal antibodies to HLA class I antigens (mean 32 pg/ml ±SD 4.5 vs control cells 53 ±SD 7.2 pg/ml). Serum EGF levels were evaluated in heart transplant recipients with and without AMR. In patients with AMR (n=16), the mean serum EGF levels were significantly reduced as compared to patients with no AMR (n=22) (151.6± SD 73 pg/mlvs.237.4± SD 52.94 pg/ml, p <0.05). Our data indicates reduced EGF expression in heart transplantation recipients with AMR. This reduction might suggest that EGF could have a protective effect in heart transplant recipients during AMR. This is the first study to demonstrate a correlation between serum EGF levels and AMR in heart transplant recipients.

The effect of endothelial progenitor cell transplantation on neointimal hyperplasia and reendothelialisation after balloon catheter injury in rat carotid arteries

BackgroundReendothelialisation is the natural pathway that inhibits neointimal hyperplasia and in-stent restenosis. Circulating endothelial progenitor cells (EPCs) derived from bone marrow (BM) might contribute to endothelial repair. However, the temporal and spatial distributions of reendothelialisation and neointimal hyperplasia after EPC transplantation in injured arteries are currently unclear.MethodsA carotid balloon injury (BI) model was established in Sprague-Dawley rats, and PKH26-labelled BM-derived EPCs were transplanted after BI. The carotid arteries were harvested on the first, fourth, seventh, and 14th day post-injury and analysed via light-sheet fluorescence microscopy and pathological staining (n = 3). EPC and human umbilical vein endothelial cell culture supernatants were collected, and blood samples were collected before and after transplantation. The paracrine effects of VEGF, IGF-1, and TGF-β1 in cell culture supernatants and serum were analysed by enzyme-linked immunosorbent assay (n = 4).ResultsTransplanted EPCs labelled with PKH26 were attached to the injured luminal surface the first day after BI. In the sham operation group, the transplanted EPCs did not adhere to the luminal surface. From the fourth day after BI, the mean fluorescence intensity of PKH26 decreased significantly. However, reendothelialisation and inhibition of neointimal hyperplasia were significantly promoted by transplanted EPCs. The degree of reendothelialisation of the EPC7d and EPC14d groups was higher than that of the BI7d and BI14d groups, and the difference in neointimal hyperplasia was observed between the EPC14d and BI14d groups. The number of endothelial cells on the luminal surface of the EPC14d group was higher than that of the BI14d group. The number of infiltrated macrophages in the injured artery decreased in the EPC transplanted groups.ConclusionsTransplanted EPCs had chemotactic enrichment and attached to the injured arterial luminal surface. Although decreasing significantly after the fourth day at the site of injury after transplantation, transplanted EPCs could still promote reendothelialisation and inhibit neointimal hyperplasia. The underlying mechanism is through paracrine cytokines and not differentiation into mature endothelial cells.

A multiplexed immuno-sensor for on-line and automated monitoring of tissue culture protein biomarkers

Frequent on-line and automated monitoring of multiple protein biomarkers level secreted in the culture media during tissue growth is essential for the successful development of Tissue Engineering and Regenerative Medicine (TERM) products. Here, we present a low-cost, rapid, reliable, and integrable anion-exchange membrane-(AEM) based multiplexed sensing platform for this application. Unlike the gold-standard manual ELISA test, incubation/wash steps are optimized for each target and precisely metered in microfluidic chips to enhance selectivity. Unlike optical detection and unreliable visual detection for the ELISA test, which require standardization for every usage, the AEM ion current signal also offers robustness, endowed by the pH and ionic strength control capability of the ion-selective membrane, such that a universal standard curve can be used to calibrate all runs. The electrical signal is enhanced by highly charged silica nanoparticle reporters, which also act as hydrodynamic shear amplifiers to enhance selectivity during wash. This AEM-based sensing platform is tested with vascular protein biomarkers, Endothelin-1 (ET-1), Angiogenin (ANG) and Placental Growth Factor (PlGF). The limit of detection and three-decade dynamic range are comparable to ELISA assay but with a significantly reduced assay time of 1 h vs 7 h, due to the elimination of calibration and blocking steps. Optimized protocol for each target renders the detection highly reliable with more than 98% confidence. The multiplexed detection capability of the platform is also demonstrated by simultaneous detection of ET-1, ANG and PlGF in 40 μl of the vascular endothelial cell culture supernatants using three-membrane AEM sensor and the performance is validated against ELISA.

ADAM10 and Src‐signalling contribute to the inflammation‐induced formation of VE‐cadherin fragments in endothelial cells

It has been shown that the release of Vascular Endothelial‐cadherin fragments (sVE‐cadherin) is closely associated with a loss of endothelial barrier function in an acute inflammation. In this context, it has been suggested that sVE‐cadherin could be a suitable marker for early detection of systemic inflammation and sepsis. However, the detailed mechanisms underlying sVE‐cadherin formation in endothelial cells remain largely unclear. Based on previous findings that suggested a critical role for the activation of the sheddase ADAM10 (a disintegrin and metalloproteinase domain‐containing protein 10), we hypothesize that ADAM10 activation might be dependent on intracellular signaling following pro‐inflammatory stimuli.Human dermal microvascular endothelial cells (HDMECs) were incubated with pro‐inflammatory cytokine Tumor necrosis factor α (TNFα), bacterial Lipopolysaccharide (LPS) from E. coli or a combination of both to mimic acute inflammation. As revealed by ELISA‐based measurements, this resulted in increased levels of sVE‐cadherin in cell culture supernatants compared to the baseline and to untreated controls. Co‐incubation of endothelial cells with the ADAM10‐inhibitor, GI254023X, together with LPS, TNFα or both significantly attenuated inflammation‐induced increase of sVE‐cadherin levels in endothelial cell culture supernatants. Measurements of Transepithelial Electrical Resistance (TER) showed a significant loss of endothelial barrier function after application of TNFα, LPS or both compared to untreated controls. Incubation of endothelial cells with the ADAM10‐inhibitor, GI254023X, together with LPS, TNFα or both significantly blocked inflammation‐induced loss of TER. In line with this inflammation‐induced intercellular gap formation, loss of VE‐cadherin at endothelial cell borders and stress fiber formation in the actin cytoskeleton were attenuated by treatment with the ADAM10‐inhibitor. As has been described previously application of TNFα, LPS or both resulted in augmented phosphorylation of tyrosinkinase src. Importantly, using cell viability assays inflammation‐induced loss of endothelial barrier function was not caused by increased cell death under the conditions used here. The selective Src inhibitor, PP2 attenuated inflammation‐induced loss of endothelial barrier function in TER measurements. Moreover, application of PP2 blocked sVE‐cadherin release, indicating a crosstalk between inflammation‐induced activation of src and ADAM10 activation.In summary, our present data confirm previous findings demonstrating that the inhibition of ADAM10 attenuates sVE‐cadherin formation after the cells were exposed to inflammatory stimuli. Furthermore, inflammation‐induced activation of src contributes to sVE‐cadherin release which suggests that ADAM10 may be regulated by src‐dependent activation in inflammation.Support or Funding InformationYalda Ghoreishi, Sven Flemming, Nicolas SchlegelDepartment of General, Visceral, Transplant, Vascular and Pediatric Surgery University Hospital Wuerzburg, Oberduerrbacherstrasse 6, 97080 Wuerzburg, Germany

Endothelial cell-derived nidogen-1 inhibits migration of SK-BR-3 breast cancer cells

BackgroundThe tumour microenvironment is a critical regulator of malignant cancer progression. While endothelial cells have been widely studied in the context of tumour angiogenesis, their role as modulators of cancer cell invasion and migration is poorly understood.MethodsWe have investigated the influence of endothelial cells on the invasive and migratory behaviour of human cancer cells in vitro.ResultsUpon exposure to culture supernatants of endothelial cells, distinct cancer cells, such as SK-BR-3 cells, showed significantly increased invasion and cell migration concomitant with changes in cell morphology and gene expression reminiscent of an epithelial-mesenchymal transition (EMT). Interestingly, the pro-migratory effect on SK-BR-3 cells was significantly enhanced by supernatants obtained from subconfluent, proliferative endothelial cells rather than from confluent, quiescent endothelial cells. Systematically comparing the supernatants of subconfluent and confluent endothelial cells by quantitative MS proteomics revealed eight candidate proteins that were secreted at significantly higher levels by confluent endothelial cells representing potential inhibitors of cancer cell migration. Among these proteins, nidogen-1 was exclusively expressed in confluent endothelial cells and was found to be necessary and sufficient for the inhibition of SK-BR-3 cell migration. Indeed, SK-BR-3 cells exposed to nidogen-1-depleted endothelial supernatants showed increased promigratory STAT3 phosphorylation along with increased cell migration. This reflects the situation of enhanced SK-BR-3 migration upon stimulation with conditioned medium from subconfluent endothelial cells with inherent absence of nidogen-1 expression.ConclusionThe identification of nidogen-1 as an endothelial-derived inhibitor of migration of distinct cancer cell types reveals a novel mechanism of endothelial control over cancer progression.

The Glycolytic Enzyme PFKFB3 Controls TNF-α-Induced Endothelial Proinflammatory Responses.

Endothelial cells play an important role in health and a variety of diseases. Recent evidences show that endothelial cells rely on glycolysis rather than on oxidative phosphorylation to generate energy to support cellular functions such as angiogenesis. However, the effect of endothelial glycolysis on vascular inflammation remains little known. Here, we investigate the role of key glycolytic enzyme PFKFB3 in tumor necrosis factor-α (TNF-α)-induced endothelial proinflammatory responses. siRNAs were used to knockdown the expression of PFKFB3. In some experiments, PFKFB3 inhibitors were also used. TNF-α at 20ng/ml was added to confluent endothelial cells for different time period of stimulation. PFKFB3 expression was examined by RT-PCR and western blotting. Cytokine antibody panel membranes were employed to detect different cytokines/chemokines in culture supernatant of endothelial cells. The determination of monocyte adhesion to endothelial cells after TNF-α treatment was conducted using THP-1 cells. The monocyte attraction was performed using Transwell filters. For further mechanisms, NF-κB-p65 localization was examined by immunofluorescence. Expression of total IκB, phospho-IκB, phospho-NF-κB-p65, and Ikkβ was detected by western blotting. DNA-binding activity of NF-κB was assessed using electrophoretic mobility shift assay. We found that TNF-α increased endothelial PFKFB3 expression. Knockdown of PFKFB3 almost blocked all TNF-α-induced release of the proinflammatory cytokines/chemokines (MCP-1, IL-8, CXCL1, GMCSF, RANTES, TNF-α) and ICAM-1. PFKFB3 knockdown also significantly inhibited TNF-α-induced monocyte adhesion and transmigration. Furthermore, inhibition of PFKFB3 inhibited TNF-α-induced Ikkβ phosphorylation, IκBα phosphorylation and degradation, NF-κB-p65 phosphorylation, nuclear translocation, and DNA-binding activity. Thus, our results demonstrate that glycolytic enzyme PFKFB3 plays a critical role in TNF-α-induced endothelial inflammation.

Effects of Yinian Jiangya Decoction (颐年降压饮) containing serum on cytokines secretion of vascular endothelium of spontaneously hypertensive rats

To observe the effects of Yinian Jiangya Decoction (YNJYD) on cytokine secretion in spontaneoulsy hypertensive rats (SHRs) vascular endothelium. Aortic endothelial cells (ECs) were primarily cultured from SHRs; male SD rats were treated with different doses (high, medium, and low doses) of YNJYD, the blood was collected on the 21st day, and then, the serum was separated. ECs were cocultured with the serum for different time courses, and the culture supernatant concentrations of endothelin (ET)-1, nitric oxide (NO), tissue-type plasminogen activator (t-PA), and plasminogen activator inhibitor (PAI-1) were determined by ABC-ELISA methods. ET-1, NO, t-PA, and PAI-1 levels in endothelial cell culture supernatant were increased in a time-dependent manner; YNJYD could significantly elevate NO and t-PA expressions in ECs, while ET-1 and PAI-1 expressions were dramatically decreased; these effects of YNJYD were in a concentration-dependent manner. The therapeutic effect of YNJYD on hypertension is attributed to its effect on regulating vessel dilation and blood coagulation, in which ET-1/NO and PAI-1/t-PA are two pairs of pivotal mediators.

Metalloproteinase 2 cleaves in vitro recombinant TRAIL: Potential implications for the decreased serum levels of TRAIL after acute myocardial infarction

BackgroundThis study was designed to evaluate the potential role of metalloproteinase 2 (MMP2) in promoting the cleavage of TNF-related apoptosis inducing ligand (TRAIL), whose circulating levels are decreased after acute myocardial infarction (AMI). MethodsThe levels of MMP2 and of tissue inhibitor of metalloprotease 2 (TIMP2), as well as of TRAIL, were measured by ELISA in the serum of AMI patients and in the culture supernatant of endothelial cells. ResultsIn AMI patients the serum levels of TRAIL showed a significant inverse correlation with the MMP2/TIMP2 ratio. In vitro MMP2 induced the cleavage of recombinant TRAIL and inactivated its ability of inducing apoptosis. Moreover, exposure of endothelial cells to TNF-α increased the MMP2/TIMP2 ratio in the culture supernatant. ConclusionsAn impaired MMP2/TIMP2 ratio might be involved in the decreased levels of circulating TRAIL observed after AMI.

Acid Sphingomyelinase Secretion by Irradiated Endothelial Cells: A Role in Intestinal Epithelial Cell Damage?

Endothelial cell (EC) response has been implicated in the induction of intestinal damage following high dose radiation exposure. Furthermore, acid sphingomyelinase (ASM), a lipid hydrolase, has been shown to contribute to the pathophysiology of lung, liver, and heart dysfunction through increased amount of extracellular ASM. However, the potential role of ASM release in radiation-induced intestinal damage has not yet been considered. Therefore, the aim of this study was to investigate whether ASM production by EC is modulated by irradiation and implicated in epithelial cell damage. First, we set up a non contact coculture model with 15 Gy-irradiated EC (HMVEC-L) and non-irradiated T84 epithelial cells to examine whether in vitro irradiation of EC resulted in the induction of epithelial cell damage via paracrine pathway. Twenty-four hours following irradiation of EC, a decreased cell number (29%) and percentage in mitosis (66%) as well as increased apoptosis (1.5-fold) and cell-surface area (1.5-fold) were shown in non-irradiated T84 cells. Next, we assessed whether ASM might be a potential mediator of this observed bystander effect. Twenty-four hours following EC radiation exposure, ASM was measured both in endothelial cell culture supernatant and lysate by ASM activity assay and Western blot. We showed for the first time that irradiation of EC induced a significant 1.4-fold increased ASM activity in culture supernatant. A 1.4-fold increased ASM was also observed in EC lysate. Then, we determined the effect of exogenous ASM on T84 cells. Human placental ASM induced a dose-dependent decreased epithelial cell number and increased apoptosis. A concentration of 100 mU/ml of exogenous ASM recapitulated the deleterious effects in T84 cells observed in bystander coculture conditions and correlated with the calculated ASM concentration in irradiated EC culture supernatant. RNA interference of ASM is currently used to determine if silencing ASM in irradiated EC reduced epithelial cell damage. Finally, we measured ASM activity in serum of 15-Gy total body irradiated mice. We showed for the first time a 1.6-fold increase in serum ASM activity from 30 min to 4 h following radiation exposure. Together our data suggest that endothelial ASM may be a key mediator in the pathogenesis of radiation-induced intestinal dysfunction.

Quantification of endothelin-1 in human umbilical venous endothelial cell culture supernatants of small for gestational age and preeclampsia neonates

Purpose : It was generally accepted now a days that the pathogenesis of preeclampsia, small for gestational age (SGA), intrauterine growth retardation and fetal origin of adult diseases were related with a endothelial cell dysfunction. The purpose of this study was to know the relation of such diseases by assessing the level of endothelin-1. Methods : SGA babies, newborns of preeclampsia and normal control mother were included in this study. Isolated endothelial cells were centrifugated and mixed with media in 37℃, 5% CO2 to obtain confluent monolayer of cultured human umbilical venous endothelial cell (HUVEC). Endothelin-1 levels were determined by Endothelin-1 colorimetric (EIA) Kits. We examined the endothelin-1 level in the HUVEC supernatants from SGA baby, and newborns from preeclampsia as well as normal mother. Also, we compared the endothelin-1 level of cultured normal HUVEC incubated with serum from cord blood of SGA, babies of preeclampsia or normal control mother. Results : The endothelin-1 levels in cultured HUVEC supernatants of three groups showed no significant difference but the endothelin-1 levels of cultured normal HUVEC incubated with serum from preeclampsia mother or SGA mother was significantly higher than those from newborns of control mothers (P

Identification and quantification of metabolites of arachidonic acid from cultures of endothelial cells by HPLC-MS2

Epoxyeicosatrienoic acids (EETs) and hydroxyeicosatetraenoic acids (HETEs) are oxidative products of arachidonic acid, some of which participate in the regulation of vascular tone. Little is known about the production of EETs and HETEs in cultures of endothelial cells. This paper reports an assay for the simultaneous quantification of isomers of EETs and HETEs from endothelial cell culture supernatants by employing solid-phase extraction and liquid chromatography-mass spectrometry. The method enabled measurement of 5,6-EET, 8,9-EET, 11,12-EET, 14,15-EET, 5-HETE, 8-HETE, 11-HETE, 12-HETE and 15-HETE. The metabolites were chromatographically separated by reversed-phase HPLC and identified by negative ESI tandem mass spectrometry and this method was used to investigate the metabolism of arachidonic acid with an endothelial cell line. For quantification, the sum of signal intensities of characteristic fragment ions was used. The detection limits for 5,6-EET and of other EET and HETE isomers were 2.0, 0.64 and 8 ng ml−1 culture medium, respectively. The precision of the method was determined with spiked culture medium (three concentrations, n = 5) and the average RSD ranged from 6.0 to 24.2%. The dynamic range was 0.6–23.5 ng ml−1 culture medium for EETs and 8.0–200 ng ml−1 for HETEs. Arachidonic acid was mainly metabolised to HETEs with product levels ranging from 59.3 to 460 ng 10−6 cells. The median of 8,9-EET and 14,15-EET was 14.5 and 17.7 ng 10−6 cells, respectively, whereas 5,6-EET and 11,12-EET were below 2 ng 10−6 cells in a 5-min incubation assay at a 30 µM arachidonic acid substrate concentration.

Antagonism between interleukin 3 and erythropoietin in mice with azidothymidine-induced anemia and in bone marrow endothelial cells.

Azidothymidine (AZT)-induced anemia in mice can be reversed by the administration of IGF-IL-3 (fusion protein of insulin-like growth factor II (IGF II) and interleukin 3). Although interleukin 3 (IL-3) and erythropoietin (EPO) are known to act synergistically on hematopoietic cell proliferation in vitro, injection of IGF-IL-3 and EPO in AZT-treated mice resulted in a reduction of red cells and an increase of plasma EPO levels as compared to animals treated with IGF-IL-3 or EPO alone. We tested the hypothesis that the antagonistic effect of IL-3 and EPO on erythroid cells may be mediated by endothelial cells. Bovine liver erythroid cells were cultured on monolayers of human bone marrow endothelial cells previously treated with EPO and IGF-IL-3. There was a significant reduction of thymidine incorporation into both erythroid and endothelial cells in cultures pre-treated with IGF-IL-3 and EPO. Endothelial cell culture supernatants separated by ultrafiltration and ultracentrifugation from cells treated with EPO and IL-3 significantly reduced thymidine incorporation into erythroid cells as compared to identical fractions obtained from the media of cells cultured with EPO alone. These results suggest that endothelial cells treated simultaneously with EPO and IL-3 have a negative effect on erythroid cell production.

Human endothelial culture supernatant selectively promotes IgM-producing hybridomas

We compared the effect of human endothelial cell culture supernatant (HECS), ESG (Ewing sarcoma growth factor), IL-6 (interleukin-6) and peritoneal macrophages on the recovery of hybridoma cells after fusion with respect to growth, stability and distribution of isotype variants. A selective growth of murine IgM-producing hybridoma cells was observed in the presence of HECS after cell fusion.

Detection of minimal but significant amount of von Willebrand factor in human omentum mesothelial cell cultures

The presence of von Willebrand factor (vWF) in human mesothelial cells is a controversial issue. The aim of this paper was to investigate the presence of vWF in human mesothelial cell cultures by means of multiple specific techniques and to compare the amount of vWF to that in endothelial cell cultures. Morphological evidence that vWF is present in the cell cytoplasm in human omentum mesothelial cells (HOMC) has been obtained by vWF staining by means of anti-vWF antibodies and immunofluorescence. The vWF concentration value (measured by ELISA) was 0.02 ng/mL in the supernatant of HOMC (160,000 ng/mL cells/mL after 12-48 hours) and between 0.08-0.12 ng/mL in the supernatant of endothelial cell cultures (160,000 cells/mL). Ethidium-bromide staining of PCR products recorded the typical vWF signal both in the endothelial and in the mesothelial cell cultures, although it was noticeably more intense in the endothelial cell culture. These data show that minimal but significant amounts of vWF are present in human mesothelial cell cultures.

Expression of tissue-type plasminogen activator, plasminogen activator inhibitor and von Willebrand factor in the supernatant of endothelial cell cultures in response to the seeding of adenocarcinoma cell line HRT-18

Plasminogen activator inhibitor (PAI-1), tissue type plasminogen activator (tPA) and von Willebrand factor (vWF) concentrations were measured by ELISA in the supernatant of the following cultures: endothelial cells from human umbilical vein (HUVEC); human colon cancer cells (HRT-18); and co-culture cells of HUVEC + HRT-18. No measurable amount of the three substances was found in the supernatant of HRT-18 cell culture. Compared to the value in the HUVEC supernatant, in the UVEC/HRT-18 co-cultures, tPA concentration was significantly lower (P = 0.0047), PAI-1 significantly higher (P = 0.026) and vWF also significantly higher (P = 0.0048). These data indicate that HRT-18 tumor cells do not produce tPA, PAI-1 and vWF; however, these tumor cells induce endothelial cells to change the production of these substances. As a consequence, the interaction between tumor and endothelial cells in vivo may lead to depression of fibrinolysis and enhancement of platelet adhesion.

The role of interferon ? in human cytomegalovirus-mediated inhibition of HLA DR induction on endothelial cells

Human cytomegalovirus (HCMV), a member of the virus family Herpesviridae that is associated with extensive worldwide morbidity and mortality in immunocompromised hosts, inhibits interferon-gamma (IFN gamma)-mediated induction of human leukocyte antigen (HLA) class II antigens on endothelial cells. In this study, the ability of HCMV-infected endothelial cells to synthesize interferon-beta (IFN beta), and the role of IFN beta in HCMV-mediated inhibition of HLA class II induction, was investigated. As determined by an encephalomyocarditis virus protection assay, HCMV-infected endothelial cell culture supernatants contained 240 IU/ml of IFN type I activity, of which 99.9% was IFN beta, as compared to the absence of IFN beta in mock-infected culture supernatants. UV-irradiated supernatants from HCMV-infected cultures inhibited induction of HLA class II in noninfected cultures by 24%. This inhibition could be abolished with 500 NU/ml of anti-IFN beta antibody. Addition of anti-IFN beta antibody directly to HCMV-infected cultures mitigated but did not abolish HLA class II antigen inhibition. Dual immunohistochemistry for HCMV and HLA DR demonstrated that infected cells, in contrast to noninfected cells, were rarely induced to express HLA class II even in the presence of anti-IFN beta antibody. These findings suggest that HCMV inhibits induction of HLA class II antigens by IFN beta dependent and independent mechanisms.

Glomerular epithelial cells produce endothelin-1.

Endothelin-1, a vasoactive peptide originally isolated from vascular endothelial cell culture supernatants, has constricting or mitogenic effects on smooth muscle and glomerular mesangial cells. Whether or not cultured rat glomerular epithelial cells synthesize endothelin-1 was assessed. Under basal culture conditions, the synthesis and release of endothelin-1 peptide by glomerular epithelial cells was time dependent, reaching 0.231 +/- 0.017 pg/1,000 cells at 24 h. For comparison, unstimulated bovine pulmonary artery endothelial cells and rat mesangial cells produced 0.982 +/- 0.237 and 0.004 +/- 0.002 pg of endothelin-1 peptide/1,000 cells per 24 h, respectively. In addition to endothelin-1 peptide, unstimulated glomerular epithelial cells expressed preproendothelin-1 mRNA. Transforming growth factor-beta, complement C5b-9, thrombin, and phorbol myristate acetate significantly enhanced endothelin-1 peptide synthesis in glomerular epithelial cells (45, 15, 55, and 25% above basal levels at 24 h, respectively), whereas epidermal growth factor had no effect. Thrombin and phorbol myristate acetate appeared to stimulate endothelin-1 peptide by activating protein kinase C, because the protein kinase inhibitor 1-(5-isoquinolinyl-sulfonyl)-3-methyl-piperazine abolished the thrombin- and phorbol myristate acetate-induced rise in endothelin-1 but had no effect on basal production. The stimulatory effect of thrombin was also markedly diminished in glomerular epithelial cells that had been depleted of protein kinase C by prolonged preincubation with a high dose of phorbol myristate acetate. Thus, glomerular epithelial cells may be an important source of endothelin-1, which might influence glomerular vasoconstriction or proliferation of target cells, particularly in the presence of proinflammatory molecules in the glomerulus.

Heparin induces release of extrinsic: Coagulation pathway inhibitor (EPI)

Extrinsic pathway inhibitor (EPI) is a potent inhibitor of the factor VIIa-tissue thromboplastin complex. The effect of heparin on EPI activity was studied using a chromogenic substrate assay. Addition of heparin to test plasma or whole blood in vitro increased EPI activity. This increase in EPI activity was reduced by the addition of polybrene and/or antibodies to anti-thrombin (anti-AT). Polybrene was therefore added to the assay system. However, part of this effect (up to 20 % of baseline value) was not abolished. After intravenous injection, EPI activity increased dose-dependently. The increase was about 200 % of baseline value after 7500 LJ heparin, and was not reduced by the addition of polybrene and/or anti-AT. A slower and prolonged increase in EPI activity occurred after subcutaneous injections of unfractionated heparin and low molecular weight heparin (LMWH). Venous occlusion failed to increase EPI activity levels. Normal EPI values were observed in patients with severe liver cirrhosis and during warfarin treatment. Gel filtration of human endothelial cell culture supernatant revealed one inhibitory fraction with molecular weight about 43.000. We conclude that EPI probably is produced in endothelial cells and may be released by heparin.

Vascular Endothelial Cells and Granulopoiesis: Interleukin-1 Stimulates Release of G-CSF and GM-CSF

AbstractCultured mononuclear phagocytes produce soluble factors that stimulate endothelial cells to release GM-colony-stimulating activity (GM-CSA). One such factor was recently identified as interleukin 1 (IL 1). Studies were designed to determine which types of granulopoietic factors are released by IL 1-stimulated endothelial cells. Supernatants from endothelial cells cultured for 3 days in medium containing IL 1 α and β were tested in both murine and human CFU-GM colony growth assays. The effect of conditioned media on differentiation of WEHI-3B myelo-monocytic leukemic cells was also examined. Control media containing IL 1 alone or unstimulated endothelial cell-conditioned media contained no detectable CSA in any bioassay. Medium conditioned by IL 1 -stimulated endothelial cells stimulated the clonal growth of both human and murine CFU-GM and induced macrophage differentiation of WEHI-3B cells. Treatment of these conditioned media with a highly specific neutralizing monoclonal G-CSF antibody completely inhibited their activity in the murine CFU-GM assay, but only partially inhibited GM colony growth by human marrow. Treatment of the active conditioned media with a neutralizing rabbit anti-human GM-CSF antibody partially reduced the activity of the media in the human GM-colony growth assay. G-CSF radioimmunoassay of endothelial cell culture supernatants and Northern blot analysis of endothelial cell cytoplasmic RNA for GM-CSF gene transcripts confirmed that IL 1 induced expression of both G-CSF and GM-CSF genes. Because treatment of media with both antibodies abrogated all activity in the human GM colony growth assay, we conclude that IL 1 -stimulated endothelial cells release both G and GM-CSF and that these are the only granulopoietic factors detectable in clonogenic assays released by these cells in vitro.

Vascular endothelial cells and granulopoiesis: interleukin-1 stimulates release of G-CSF and GM-CSF

Cultured mononuclear phagocytes produce soluble factors that stimulate endothelial cells to release GM-colony-stimulating activity (GM-CSA). One such factor was recently identified as interleukin 1 (IL 1). Studies were designed to determine which types of granulopoietic factors are released by IL 1-stimulated endothelial cells. Supernatants from endothelial cells cultured for 3 days in medium containing IL 1 alpha and beta were tested in both murine and human CFU-GM colony growth assays. The effect of conditioned media on differentiation of WEHI-3B myelomonocytic leukemic cells was also examined. Control media containing IL 1 alone or unstimulated endothelial cell-conditioned media contained no detectable CSA in any bioassay. Medium conditioned by IL 1-stimulated endothelial cells stimulated the clonal growth of both human and murine CFU-GM and induced macrophage differentiation of WEHI-3B cells. Treatment of these conditioned media with a highly specific neutralizing monoclonal G-CSF antibody completely inhibited their activity in the murine CFU-GM assay, but only partially inhibited GM colony growth by human marrow. Treatment of the active conditioned media with a neutralizing rabbit anti-human GM-CSF antibody partially reduced the activity of the media in the human GM-colony growth assay. G-CSF radioimmunoassay of endothelial cell culture supernatants and Northern blot analysis of endothelial cell cytoplasmic RNA for GM-CSF gene transcripts confirmed that IL 1 induced expression of both G-CSF and GM-CSF genes. Because treatment of media with both antibodies abrogated all activity in the human GM colony growth assay, we conclude that IL 1-stimulated endothelial cells release both G and GM-CSF and that these are the only granulopoietic factors detectable in clonogenic assays released by these cells in vitro.