Differences in coagulant and fibrinolytic activities of cultured human endothelial cells derived from omental tissue microvessels and umbilical veins

Large vessel and microvascular endothelial cells were compared in their capacity to synthesize and secrete coagulant and fibrinolytic factors. Human omental tissue microvascular endothelial cells (HOTMEC) and human umbilical vein endothelial cells (HUVEC) were isolated, grown to confluency under identical conditions, and studied in primary cultures. After an incubation period of 12 hours in serum-free medium, the conditioned medium of confluent HOTMEC contained 100-fold higher levels of tissue plasminogen activator (tPA) antigen than that of HUVEC. The conditioned media as well as the lysates of both cell types did not contain any free tPA activity, but the free plasminogen activator inhibitor capacity was found intracellularly as well as extracellularly. Although von Willebrand factor was detected in both cell types by immunofluorescence, measurable amounts were only found in HUVEC using an enzyme-linked immunosorbent assay. The kinetics of protein C activation by thrombin on the surface of once-passaged cells were identical for HOTMEC and HUVEC. The present study indicates that cultivated HOTMEC produce larger quantities of tPA than HUVEC do, possess smaller amounts of von Willebrand factor than HUVEC do, and express thrombomodulin for protein C activation as effectively as HUVEC.     

Synthesis of von Willebrand Factor by Cultured Human Endothelial Cells

Cultured human endothelial cells synthesize and secrete a protein(s) which has Factor VIII antigen but which lacks Factor VIII clot-promoting activity (J. Clin. Invest. 52, 2757-2764, 1973). Von Willebrand factor activity has been identified in medium from cultured human endothelial cells. This activity was demonstrated by the ability to correct the defect in platelet adhesiveness of blood obtained from patients with von Willebrand's disease. This activity also supported ristocetin-induced aggregation of washed normal human platelets. The von Willebrand factor activity from cultured endothelial cells has physicochemical and immunologic properties like those of the von Willebrand factor activity and the Factor VIII antigen present in human plasma and the Factor VIII antigen synthesized by human endothelial cells in vitro. Rabbit antibody to chromatographic fractions containing endothelial cell von Willebrand factor inhibits the platelet retention of normal blood in glass bead columns.     

Hepatocytes may produce laminin in fibrotic liver and in primary culture

Previous studies have shown that laminin is present in basement membranes in normal liver but failed to identify cellular sources. We have investigated the extracellular and intracellular distribution of laminin in normal rat and human liver, in fibrotic human liver and in primary hepatocyte cultures from both species by light and electron microscopy using the indirect immunoperoxidase technique. In normal liver from both species, antibodies to laminin strongly stained basement membranes and formed discontinuous discrete deposits in the wall of the sinusoid. Vascular endothelial and bile duct cells as well as fat-storing cells and sinusoidal endothelial cells strongly stained for this glycoprotein while hepatocytes were negative. In fibrotic human liver, increased amounts of extracellular laminin were usually found. Continuous deposition in the space of Disse was observed in some cases. In addition to fat-storing cells and endothelial cells, hepatocytes were also sometimes positive. Normal rat and human hepatocytes synthesize and secrete laminin in conventional culture, but it remains soluble in the medium. By contrast, in coculture with another rat liver cell type, laminin accumulated around hepatocyte cords. These observations suggest that fat-storing cells and endothelial cells are the major sites of production of laminin in normal liver. However, when their environment is altered (e.g., liver injury, culture), adult hepatocytes are able to synthesize detectable amounts of laminin.     

Cell types involved in collagen and fibronectin production in normal and fibrotic human liver

Three collagen types (I, III and IV) and fibronectin were localized in normal and alcoholic human liver by light and electron microscopy using the indirect immunoperoxidase technique. In normal liver, most of the bundles of collagen fibers stained for type pro-III collagen while only a few reacted for type I. Basement membranes stained for type IV collagen which formed discontinuous discrete deposits in sinusoids. Only fibronectin appeared as an almost continuous layer in the space of Disse. At the intracellular level, hepatocytes were found to contain little type I collagen and large amounts of fibronectin. Fat-storing cells strongly stained for type IV collagen and expressed low amounts of types I and III collagen and fibronectin. Endothelial cells contained low amounts of all the components. Alcoholic livers were studied at three stages: steatosis, fibrosis and cirrhosis. Qualitative and quantitative differences were observed in extracellular and intracellular distributions of matrix proteins. Increased amounts of all components were usually found in fibrotic and cirrhotic livers compared to normal liver. In two fibrotic livers which contained numerous bundles of collagen in the sinusoids, fat-storing cells stained more intensely for type III collagen. In a cryptogenic fibrotic liver, abundant type IV collagen was observed in hepatocytes. These results suggest that hepatocytes, fat-storing cells and endothelial cells are engaged in production of extracellular matrix components in normal human liver. In fibrosis, hepatocytes which normally did not synthesize types III and IV collagen may produce these collagens.     

The lipoprotein-associated coagulation inhibitor that inhibits the factor VII-tissue factor complex also inhibits factor Xa: insight into its possible mechanism of action

Blood coagulation is initiated when plasma factor VII(a) binds to its essential cofactor tissue factor (TF) and proteolytically activates factors X and IX. Progressive inhibition of TF activity occurs upon its addition to plasma. This process is reversible and requires the presence of VII(a), catalytically active Xa, Ca2+, and another component that appears to be associated with the lipoproteins in plasma, a lipoprotein-associated coagulation inhibitor (LACI). A protein, LACI(HG2), possessing the same inhibitory properties as LACI, has recently been isolated from the conditioned media of cultured human liver cells (HepG2). Rabbit antisera raised against a synthetic peptide based on the N-terminal sequence of LACI(HG2) and purified IgG from a rabbit immunized with intact LACI(HG2) inhibit the LACI activity in human serum. In a reaction mixture containing VIIa, Xa, Ca2+, and purified LACI(HG2), the apparent half-life (t1/2) for TF activity was 20 seconds. The presence of heparin accelerated the initial rate of inhibition threefold. Antithrombin III alpha alone had no effect, but antithrombin III alpha with heparin abrogated the TF inhibition. LACI(HG2) also inhibited Xa with an apparent t1/2 of 50 seconds. Heparin enhanced the rate of Xa inhibition 2.5-fold, whereas phospholipids and Ca2+ slowed the reaction 2.5-fold. Xa inhibition was demonstrable with both chromogenic substrate (S-2222) and bioassays, but no complex between Xa and LACI(HG2) could be visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Nondenaturing PAGE, however, showed that LACI(HG2) bound to Xa but not to X or Xa inactivated by diisopropyl fluorophosphate. Thus, LACI(HG2) appears to bind to Xa at or near its active site. Bovine factor Xa lacking its gamma-carboxyglutamic acid-containing domain, BXa(-GD), through treatment with alpha-chymotrypsin, was used to further investigate the Xa requirement for VIIa/TF inhibition by LACI(HG2). LACI(HG2) bound to BXa(-GD) and inhibited its catalytic activity against a small molecular substrate (Spectrozyme Xa), though at a rate approximately sevenfold slower than native BXa. Preincubation of LACI(HG2) with saturating concentrations of BXa(-GD) markedly retarded the subsequent inhibition of BXa. The VII(a)/TF complex was not inhibited by LACI(HG2) in the presence of BXa(-GD), and further, preincubation of LACI(HG2) with BXa(-GD) slowed the inhibition of VIIa/TF after the addition of native Xa. The results are consistent with the hypothesis that inhibition of VII(a)/TF involves the formation of a VIIa-TF-XA-LACI complex that requires the GD of XA.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Monocytes stimulate fibroblastoid bone marrow stromal cells to produce multilineage hematopoietic growth factors

In previous studies we have found that monocytes produce soluble factors that stimulate human umbilical vein endothelial cells to produce granulocyte-macrophage colony-stimulating activity (CSA), burst- promoting activity (BPA), and megakaryocyte colony-stimulating activity (Meg-CSA) as well as factors that stimulate T lymphocytes and neonatal fibroblasts to produce CSA. To test the hypothesis that monocytes would similarly stimulate the production of hematopoietic growth factors by autologous bone marrow stromal cells, multiply-passaged adherent fibroblastoid cells derived from the bone marrow of normal volunteers were exposed to conditioned media prepared by incubating autologous peripheral blood monocytes in complete medium for three days. When conditioned media from stromal cells incubated in monocyte-conditioned medium were compared with those of stromal cells cultured in the absence of monocyte-conditioned medium, BPA was increased fourfold and CSA was increased more than 30-fold. We conclude that mononuclear phagocytes recruit stromal cells of the marrow to produce multilineage growth factors in vitro. We suggest that these monocyte-derived recruiting activities may play an important role in orchestration of hematopoietic growth factor production by cells of the marrow microenvironment.     

Properties of rheumatoid and normal synovial tissue in vitro and cells derived from them

Summary Using organ and cell culture techniques for tissues and cells derived from human sources, we have investigated cellular interactions involving synovial tissue. Normal synovium in culture produced less prostaglandin E (PGE) and collagenase than cultures of rheumatoid synovial fragments. When synovial tissue was dissociated by enzymatic digestion, monolayers of adherent cells were established in primary culture. The adherent cells rapidly lost the ability to synthesize large amounts of PGE and collagenase and rheumatoid synovial cells became indistinguishable from normal synovial cells. Supernatants from cultured human mononuclear blood cells contained activities (Mononuclear cell factor(s)=MCF) which stimulated PGE and collagenase production by either normal or rheumatoid synovial cells. Conditioned medium from cultures of either normal or rheumatoid synovial fragments (Synovial factor(s)=SF) also stimulated production of PGE and collagenase by these human cells. Both MCF and SF also stimulated the production of PGE by cells isolated from human trabecular bone. Since both normal and rheumatoid synovial cells respond similarly to these factors, there appears to be little specificity with regard to whether the target cells are derived from normal or pathological sources. Furthermore, since both normal and rheumatoid synovium are able to produce similar amounts of stimulatory activity, inflammatory cells are not solely responsible for these phenomena. Normal synovium must therefore contain cells which can be recruited to participate in these potential cellular interactions. Destruction of joint structures may be mediated by factors of the type studied here, which may be produced when there is failure of the mechanisms that prevent them from being synthesised or released.     

Fibrinolytic properties of a human endothelial hybrid cell line (Ea.hy 926)

The fibrinolytic characteristics of the endothelial hybrid cell line EA.hy 926, established by fusing a human umbilical vein endothelial cell with a human carcinoma cell line, were studied. The hybrid cell line produced large amounts of tissue-type plasminogen activator (t- PA), plasminogen activator inhibitor type 1, and a small amount of urokinase. All plasminogen activator present in conditioned medium was complexed with inhibitor because the cells secreted plasminogen activator inhibitor in excess over plasminogen activator and no activator activity was detectable in conditioned media by direct activity assays. t-PA activator activity was, however, demonstrable in conditioned media after treatment with sodium dodecyl sulfate, in agreement with t-PA antigen determinations. Increased plasminogen activator inhibitor activity could be induced by incubating the cells in the presence of endotoxin or microtubule inhibitors, whereas increased t-PA activity could be induced by microtubule inhibitors. Interleukin-1 had no effect. The fibrinolytic characteristics of the hybrid cell line were stable for at least 30 passages. The perpetual human hybrid cell line EA.hy 926 therefore may be a useful tool for the study of fibrinolysis in cultured endothelial cells.     

Potential attenuation of fibrinolysis by growth factors released from platelets and their pharmacologic implications

Increased concentrations of the fast-acting tissue-type plasminogen activator (t-PA) inhibitor attenuate the fibrinolytic activity of pharmacologically administered activators of the fibrinolytic system such as t-PA. Accordingly, it was hypothesized that augmentation of synthesis and elaboration of inhibitor from the liver, leading to increased concentrations of inhibitor in plasma, or from endothelial cells in the vicinity of thrombi undergoing lysis, leading to increased concentrations locally, may contribute to failure of pharmacologically induced thrombolysis or to early reocclusion. Because platelets are rich in transforming growth factor beta and epidermal growth factor-like activity, it was thought that release of growth factors from platelets activated in vivo could mediate increases of the inhibitor in plasma by stimulating its formation in the liver and its local release from endothelial cells in the vicinity of thrombi. If so, fibrinolysis might be rendered more effective by concomitant prevention of platelet growth factor release. Transforming growth factor beta, a major constituent of platelets, increased concentrations of the t-PA inhibitor messenger ribonucleic acid (mRNA) in human hepatoma cells in a specific and dose-dependent manner. A peak effect was seen with 5 ng/ml and a 10-fold increase in 6 hours. Release of inhibitor protein into conditioned media increased as well. Induction of the inhibitor mRNA increase was elicited by exposure as brief as 30 minutes. Cycloheximide, an inhibitor of protein synthesis, was not inhibitory. The mechanisms responsible differed from those seen with epidermal growth factor, shown previously in the laboratory to increase inhibitor mRNA. In addition, the 2 factors were synergistic. Platelet lysates elicited effects simulating those of the purified growth factors.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Immunological Localization of Factor V in Human Tissue

Antibody to factor V was produced by immunizing rabbits with purified factor V from human plasma. Various tissues were examined for the presence of factor-V antigen using this antiserum. It was consistently demonstrated in homogenates of liver and spleen by means of an antibody (coagulation inhibitor) neutralization technique. The antigen was further localized histologically by the indirect fluorescent antiglobulin technique. It was present on the endothelium of normal blood vessels in all organs examined. In the liver it was detected in hepatic parenchymal cells and a distinctive pattern of fluorescence in the spleen suggested that it was being detected on platelets. Results were negative in all other tissues examined. The findings confirm the presence of factor V in hepatic parenchymal cells and support the suggestion that endothelial coagulation factors may play a role in haemostasis and thrombosis.     

Production of platelet-activating factor by human vascular endothelial cells: evidence for a requirement for specific agonists and modulation by prostacyclin

Primary cultures of confluent endothelial cells derived from human umbilical veins produce platelet-activating factor (PAF) (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) when stimulated with appropriate agonists. Highly purified human thrombin and calcium ionophore A23187 stimulate the incorporation of [3H] acetate into a lipid product that has been identified as PAF by its behavior in thin-layer chromatographic and high-performance liquid chromatographic systems, the presence of characteristic biologic activity, and appropriate response to phospholipases. A number of other humoral mediators, examined because they directly influence the activity of vascular cells or because they may mediate endothelial injury, do not stimulate PAF production by endothelial cells. This indicates that the synthesis of PAF by cultured human endothelial cells is a response to specific agonists and is not an unregulated event that occurs as a result of nonspecific cellular perturbation. The PAF produced by thrombin-treated endothelial cells is a potent stimulus for platelet activation, as assayed by the aggregation of human platelets in autologous plasma. The production of PAF by endothelial monolayers is attenuated by prostacyclin, another product of stimulated endothelial cells. Conversely, PAF production is enhanced by treatment of the endothelial cells with indomethacin, an inhibitor of prostacyclin synthesis from arachidonic acid, indicating that endogenously generated prostacyclin may modulate PAF synthesis. The potential to synthesize PAF, a unique lipid autocoid that stimulates the activation of both platelets and polymorphonuclear leukocytes, suggests that endothelial cells can directly influence the activity of these circulating effector cells. This biologic potential may be important in the interaction of the endothelium with circulating blood cells in physiologic conditions and in syndromes of vascular injury.     

Endothelium and endogenous fibrinolysis

Reduced activity of the endogenous fibrinolytic system contributes to intramural deposition of microthrombi in atherogenesis and to intraluminal deposition of thrombi leading to acute complications of atherosclerosis such as acute coronary syndromes. Endogenous fibrinolytic activity is predominantly regulated by the plasminogen activator inhibitor type 1 (PAI-1). Increased activity of PAI-1 leading to reduced endogenous fibrinolytic activity has been identified as an important independent risk factor for cardiovascular disease. Vascular endothelial cells form a barrier between the circulating blood with its dynamic balance between ongoing thrombosis and fibrinolysis and the subendothelial layers of the vascular wall with their prothrombotic activity. In addition, endothelial cells synthesize and secrete substantial amounts of plasminogen activators and their inhibitor PAI-1. Thus, endothelium plays an important role in the regulation of endogenous fibrinolysis. After describing the components of the endogenous fibrinolytic system and its interactions, this review focuses on the impact on endogenous fibrinolysis by the renin angiotensin system, the kallikrein kinin system, and type 2 diabetes mellitus. Investigations using transgenic and knock-out animal models--the results of which are also summarized--have improved our understanding of the interaction between endogenous fibrinolysis and endothelium. In each section of the review therapeutic implications and potentials are discussed.     

Human dermal mast cells contain and release tumor necrosis factor alpha, which induces endothelial leukocyte adhesion molecule 1.

Tumor necrosis factor alpha (TNF-alpha) is a proinflammatory cytokine that mediates endothelial leukocyte interactions by inducing expression of adhesion molecules. In this report, we demonstrate that human dermal mast cells contain sizeable stores of immunoreactive and biologically active TNF-alpha within granules, which can be released rapidly into the extracellular space upon degranulation. Among normal human dermal cells, mast cells are the predominant cell type that expresses both TNF-alpha protein and TNF-alpha mRNA. Moreover, induction of endothelial leukocyte adhesion molecule 1 expression is a direct consequence of release of mast cell-derived TNF-alpha. These findings establish a role for human mast cells as "gatekeepers" of the dermal microvasculature and indicate that mast cell products other than vasoactive amines influence endothelium in a proinflammatory fashion.     

Induction of endothelial cell expression of the plasminogen activator inhibitor type 1 gene by thrombosis in vivo.

BACKGROUND. We have shown previously that products from activated platelets can augment synthesis of plasminogen activator inhibitor type 1 (PAI-1) in cultured endothelial and hepatoma (Hep G2) cells in vitro and increase plasma PAI-1 activity in vivo in rabbits. Accordingly, the effects of activation of platelets associated with thrombosis and thrombolysis in vivo on plasma PAI-1 activity and expression of the PAI-1 gene in endothelium, liver, and other organs were characterized. METHODS AND RESULTS. Endothelial injury giving rise to platelet-rich thrombi was induced with electrical stimulation in carotid arteries in rabbits. Clot lysis and recanalization were induced subsequently with intravenous tissue-type plasminogen activator (t-PA) and verified with Doppler flow probes. Plasma PAI-1 activity (mean +/- SD) increased from 6 +/- 2 arbitrary units (AU)/ml to 129 +/- 48 AU/ml (n = 15) within several hours after recanalization. When t-PA had failed to induce recanalization, the increase was much less (from 7 +/- 2 to 42 +/- 23 AU/ml, n = 11). To define mechanisms responsible for these changes, PAI-1 messenger RNA (mRNA) was evaluated by Northern blot analysis and localized in tissues by in situ hybridization. Strong and consistent induction of PAI-1 mRNA was evident in aorta, heart, and liver of animals subjected to thrombosis (twofold to threefold increases compared with values in controls), particularly in those in which thrombolysis had been induced (fourfold to sixfold). After thrombolysis, an intense, PAI-1 mRNA-specific signal was detected in endothelium of aorta, liver, and heart, with less intense signals in endothelium of lung, adrenals, and kidneys. CONCLUSIONS. The increases in plasma PAI-1 activity follow a preceding increase in endothelial cell expression of the PAI-1 gene as reflected by PAI-1 mRNA levels. Thus, increased synthesis of endothelial cell PAI-1 after thrombosis and thrombolysis may attenuate endogenous fibrinolysis early after coronary thrombolysis, thereby potentiating early, thrombotic reocclusion.     

Induction of granulocyte and granulocyte-macrophage colony-stimulating factors from human monocytes stimulated by Fc fragments of human IgG

The effect of human IgG on human haemopoiesis has been studied in vitro. Dialysed purified IgG stimulated haemopoietic colony growth by bone marrow mononuclear cells (MNC) but not by monocyte-depleted MNC. Culture media, conditioned by IgG-stimulated peripheral blood MNC, augmented formation of neutrophil-macrophage, eosinophil, and megakaryocyte colonies by monocyte-depleted marrow MNC. Serum-free IgG-conditioned media also contained colony-stimulating activity (CSA). IgG Fc fragments and heat-aggregated IgG promoted the secretion of CSA, but F(ab')2 fragments, Fab fragments or ultracentrifuged IgG did not. In the cell-selection studies, CSA was produced by highly enriched monocytes following stimulation with Fc fragments. The antiserum against human granulocyte colony-stimulating factor (G-CSF) and/or granulocyte-macrophage CSF (GM-CSF) neutralized the CSA produced by Fc fragment-activated monocytes. Enzyme immunoassays showed G-CSF and GM-CSF in media conditioned by monocytes stimulated with the Fc fragments, heat-aggregated IgG and anti-D-sensitized red blood cells (RBC). Northern hybridization analysis showed mRNA encoding G-CSF and GM-CSF in RNA extracted from MNC and monocytes cultured with the Fc fragments, but not in the RNA from unstimulated cells or monocyte-depleted MNC. These results indicate that IgG Fc fragments, aggregated IgG and antigen-antibody complexes induce monocytes to produce G-CSF and GM-CSF in vitro. The CSFs release induced by IgG may be involved in the in vivo regulatory network in haemopoiesis.