Testican in human blood.

Testican is a highly conserved, differentially expressed gene product of unknown function. Since testican is expressed by human endothelial cells and includes a signal sequence, it was our hypothesis that testican protein would be present in blood. We have developed chicken antibodies specific for testican sequence near the N-terminal and identified a 130-kDa form of testican in human plasma. This is much larger than the calculated molecular weight of the encoded polypeptide, suggesting glycosylation of this plasma protein, and large forms of recombinant testican produced in culture were found to include chondroitin sulfate. The 130-kDa form of testican is unstable in plasma. It is converted to smaller stable forms by separable plasma factors that can be blocked by certain serine protease inhibitors. Testican size conversion may be important in its functional activation or decay. One testican domain has strong homology to thyropin-type cysteine protease-inhibitors. Thus, testican may have a function related to protease inhibition in the blood.

Distribution of testican expression in human brain.

Testican is a putative extracellular heparan/ chondroitin sulfate proteoglycan of unknown function that is expressed in a variety of human tissues at widely different levels but is most abundant in the brain. In mice, testican mRNA has been detected only in brain and it is therefore likely to have an important function in the central nervous system. RNA blot analysis reveals the relative intensity of testican in various regions of the human brain. Levels of testican message are most pronounced in the thalamus, hippocampus, occipital lobe, nucleus accumbens, temporal lobe, and caudate nucleus, with somewhat lower levels in the cerebral cortex, medulla oblongata, frontal lobe, amygdala, putamen, spinal cord, substantia nigra, and cerebellum. In situ hybridization reveals the cellular distribution of the mRNA within these areas to be highest in neurons and in choroid plexus epithelium, and moderately lower in ependymal cells lining the ventricles and in vascular endothelial cells. Testican mRNA is not detected in oligodendrocytes or in most astrocytes. However, astrocytes in regions of reactive gliosis do express testican mRNA. These findings, along with a cysteine-rich pattern similarity to neurocan, brevican, versican, and other proteoglycans found in brain, suggest that testican may be a part of the specialized extracellular matrix of the brain.

Efficient gene transfer to human endothelial cells using DNA complexed to adenovirus particles.

Human endothelial cells have been found to be relatively refractory to various methods of DNA transfection currently in common use. By using a transfection method involving DNA complexed with replication-deficient adenovirus particles, we have shown that 20% of a population of cultured endothelial cells can be transfected and high levels of transient expression achieved. Both early-passage human umbilical vein endothelial cells and the continuous differentiated line of human endothelium-derived EA.hy926 cells are responsive to this method of transfection. Efficient DNA transfection of endothelial cells is important for studies of endothelium-specific promoters and is a potentially useful route for transgenic therapy.

Role of ICE-like proteases in endothelial cell hypoxic and reperfusion injury.

Because of its location between blood and tissue, the endothelium is particularly vulnerable to hypoxic/reperfusion injury, but the mechanisms responsible for this injury are not known. A number of recent findings suggest that hypoxia and reperfusion injures neuronal cells via apoptosis. Apoptosis has recently been shown to depend on the activation of a class of proteases with homology to Interleukin-1 beta converting enzyme (ICE) protease. Therefore, we examined the effect of specific inhibitors of ICE-like proteases on hypoxic and reperfusion injury in cultured EAhy926 endothelial cells. Pretreatment of cells with ICE inhibitor II (Ac-YVAD-CMK), ICE inhibitor III (Z-Asp-2,6-dichlorobenzoyloxy-methylketone-Z-Asp-CH2-DCB+ ++), or ICE inhibitor IV (Ac-YVKD-CHO) (all at 10-100 microM) did not protect cells from hypoxic injury. However, pretreatment of cells with ICE inhibitor III and to a lesser extent with ICE inhibitor II, but not with ICE inhibitor IV, protected cells from reperfusion injury. The protective effect of ICE inhibitor III was not dependent upon pH, but was associated with decreased release of arachidonic acid from cells. These findings suggest that reperfusion injury to EAhy926 endothelial cells involves ICE-like proteases. The identity of the protease(s) is not known but it does not appear to be a YAMA-type protease based upon ICE inhibitor specificity. Our data also indicate that a potential target of this protease is phospholipase A2 (PLA2).

Endothelial cell expression of testican mRNA.

By screening random cDNAs from a continuous vascular endothelial cell line, EA.hy926, we identified a 5 kb mRNA that is expressed at high levels by this human cell line and by an early passage umbilical vein endothelial cell line. It is detected at lower levels in certain stromal cell lines, but it is not detected in most other cell lines tested, indicating that it represents a differentially expressed function rather than a ubiquitous or housekeeping function. This mRNA was readily detected in samples derived from most human organs as might be expected for a gene expressed in the vascular wall. Sequencing of the 5 kb mRNA reveals its identity with 3.5 kb of previously published testis-derived cDNA sequence called testican (Alliel et al., 1993). Differential expression of this gene by endothelial cells contributes a new perspective on the potential function of testican.

Anti-vascular endothelial cell antibodies (AECA): comparison of two assay methods and clinical applications.

Vascular endothelial cells may be a target for autoantibodies (AECAs) against membrane antigens that are constitutively expressed, induced or bound to their surface. To test this hypothesis, we used an enzyme-linked immunosorbent assay (ELISA) with two types of human endothelial cells as the substrate, i.e., human umbilical cord vein endothelial cells (HUVECs) or the hybrid cell line EAhy-926 obtained by fusion of HUVECs with the bronchial carcinoma cell line A549. A comparative functional study of these two cell types demonstrated that EAhy-926 cells produced only small amounts of VIII von Willebrand factor and tissular factor, did not contain Weibel Palade bodies visible under the electron microscope, and expressed ICAM-1 and selectin E in levels of no more than 15% of those expressed by human umbilical cord vein endothelial cells both after stimulation by bacterial lipopolysaccharide and under basal conditions. However, the two assay methods yielded similar IgG AECA titers when used on sera from patients with rheumatoid vasculitis or antiphospholipid syndrome. These antibodies did not exhibit cytotoxicity for cord vein or EAhy-926 cells. They were not specific for endothelium, since their activity decreased by a mean of 40% after incubation of sera with the epithelial cell line A549. A cross-sectional study of 565 sera demonstrated that anti-vascular IgG and IgM AECAs reactive with EAhy-926 cells occurred mainly in patients with dermatomyositis (IgG, 58%; IgM, 22%), systemic scleroderma (IgG, 48%; IgM, 18%), primary Sjögren's syndrome (IgG, 44%; IgM, 12%) and secondary and primary systemic vasculitides (IgG, 38%; IgM, 18%) including Wegener's granulomatosis. A longitudinal study in patients with Wegener's granulomatosis showed that AECAS were predictive of disease activity.

Tumor cell--endothelium adhesion in an artificial venule.

Adhesion of tumor cells to endothelial cells is a crucial step in the complex sequence of metastasis. In addition to type and local density of adhesion molecules on both cell types, shear forces exerted by the blood flow have been described to be of major importance in governing cell adhesion. Most of the experiments on the molecular basis of tumor-endothelial cell adhesion have been performed as static assays which lack shear forces. We have developed an artificial venule which shares the following in vivo characteristics. A confluent layer of endothelial cells lines the luminal surface of a glass capillary of 1 mm i.d. with pores of 30 nm diameter to allow diffusion of molecules from outside the capillary. Physiological pressure of 16 mbar, flow rate of 2 cm/s, and shear forces of 2 dynes/cm2 are maintained. This device allowed us to show that under dynamic conditions adhesion of B16 mouse melanoma cells to EA.hy926 endothelial cells is mediated most likely by a lectin-like structure on B16 cells and oligosaccharide(s) on endothelial cells. In addition, endothelial activation-independent adhesion was found to be restricted to only a fraction of endothelial cells, as the number of B16 cells that adhered was independent of the number of B16 cells applied.

Tetrahydrobiopterin as another EDRF in man.

Endotoxin and inflammatory cytokines downregulate expression of constitutive nitric oxide synthase (cNOS) in human vascular endothelial cells with concomitant increase of tetrahydrobiopterin synthesis in these cells and parallel upregulation of inducible NOS expression in smooth muscle cells, indicating compartmentalized nitric oxide (NO) production under septic conditions in man. In this report the compartmentalization has been further studied using dual chamber cell cultures with inflammatory activated human endothelial cells. We show that endothelial cells secrete BH4 vectorially into the basal direction thereby providing underlining smooth muscle cells with the cofactor necessary for NO production. Furthermore, by laser Doppler velocimetry we show that intraarterial infusion of BH4 induces strong vasodilatation in man. Consumption of L-arginine and production of cyclic GMP increased and therefore imply NO as second messenger. Thus the discovery of an endothelium-derived factor regulating NOS activity would reconcile the concept of an inflammatory EDRF that is not NO itself but results in NO-dependent vasodilatation in man.

Modulation of human endothelial cell tetrahydrobiopterin synthesis by activating and deactivating cytokines: new perspectives on endothelium-derived relaxing factor.

Endothelial cells, through soluble mediators, play an important role in the regulation of the vascular tone. In the present paper we investigated whether endothelial tetrahydrobiopterin (BH4), an obligatory cofactor of nitric oxide (NO) synthase, could serve as such a regulatory mediator. By studying the human vascular endothelial hybrid cells EA hy 926, we found that 1) BH4 biosynthesis is highly regulated (70-fold) by activating and deactivating cytokines; that 2) up to 90% of the induced BH4 is released by activated endothelium, and 3) while intracellular BH4 could be related to cyclic GMP concentrations within the endothelial cells, the bulk of BH4 (up to 90 pmol/10(6) cells) appears not to serve endothelial cell requirements. Activation and deactivation of BH4 synthesis by cytokines was paralleled by other endothelial cell responses reflecting their activity. We propose that BH4 serves as an endothelial mediator augmenting the activity of cytokine-inducible NO synthase in vascular smooth muscle cells. BH4 could thereby account for endothelium-derived relaxing factor activity.

Application of an immortalized human endothelial cell line to the leucocyte:endothelial adherence assay.

This report shows that an immortalized endothelial cell line (EA.hy 926) is able to substitute for secondary cultures of human umbilical vein endothelial cells (HUVEC) in the leucocyte:endothelial adherence assay. Enriched preparations of blood polymorphonuclear leucocytes, monocytes and resting and activated lymphocytes exhibited similar adherence characteristics to HUVEC and the EA.hy 926 cells. Cytokines such as tumour necrosis factor (TNF) act on endothelial cells to increase their adhesiveness for leucocytes and in this study there was no difference between TNF-treated HUVEC and EA.hy 926 cells in supporting the enhanced binding of leucocytes. The adherence promoting effect of TNF-treated EA.hy 926 cells appears to be dependent upon their endothelial properties since TNF treatment of A549 cells, the permanent human cell line used to generate the hybrid EA.hy 926 cells did not augment lymphocyte attachment. Monoclonal antibodies against CD11a and CD18 inhibited the binding of lymphocytes to untreated and TNF-treated HUVEC and EA.hy 926 cells and ICAM-1 expression was increased on both monolayers following treatment with TNF. The availability of a hybrid endothelial cell line whose adhesive properties are similar to those of recently isolated endothelial cells should benefit the study of factors that govern leucocyte-endothelial cell interactions and be advantageous to the longitudinal investigation of leucocyte adherence under static conditions.

Extent of differentiated gene expression in the human endothelium-derived EA.hy926 cell line.

Vascular endothelial cells perform many differentiated functions in processes such as angiogenesis, hemostasis, and inflammation. The number of recognized differentiated functions has increased rapidly in recent years, but there may be many more still unrecognized. The purpose of this study is to estimate the fraction of differentially expressed mRNA in a continuous human endothelium-derived cell line, EA.hy926. Random cDNA clones representing mRNAs from this cell line were labeled and used to probe blots of RNA from EA.hy926 cells and from cells of a relatively undifferentiated line. Of 49 random cDNAs, 5 cDNAs or 10% were found to represent mRNAs that are differentially expressed in EA.hy926 and in early passage umbilical vein endothelial cells. Since more than 10(4) different genes are thought to be expressed in the typical mammalian cell, our data indicate that about 10(3) gene products contribute to the differentiated properties of endothelial cells.

In vitro model of angiogenesis using a human endothelium-derived permanent cell line: contributions of induced gene expression, G-proteins, and integrins.

The EA hy926 cell line is a continuous, clonable, human cell line that displays a number of features characteristic of vascular endothelial cells (Edgell et al., 1983). Here we report that when EA hy926 cells (EA cells) are plated on an extracellular matrix material [Matrigel], they undergo a process of morphological re-organization leading to the formation of a complex network of cord or tubelike structures. These events seem to resemble, in some respects, an in vitro process of angiogenesis. The morphological re-arrangement occurs within a 12-16 hr period and seems to require expression of new messenger RNA and protein, since it is completely blocked when actinomycin D or cycloheximide are present at the time the cells are plated on Matrigel. This is not due to overt toxicity of the drugs, since exposure of cells to actinomycin D at 2 hr or more after plating on Matrigel has little effect on the formation of the tubelike structures. The process of Matrigel-induced tube formation also apparently involves a G-protein mediated signal. Treatment of the EA cells with pertussis toxin completely blocks the process and causes the ADP-ribosylation of a 42 kD protein that is recognized by an antibody to Gi-alpha subunits. In contrast, concentrations of pertussis toxin sufficient to block tube formation have only modest effects on the adhesion or motility of EA cells on purified matrix components such as laminin or collagen IV. The process of Matrigel-induced tube formation also involves integrins since monoclonal antibodies to integrin alpha 6 or beta 1 subunits can completely block the process. The concentrations of anti-integrin antibodies needed to block tube formation are much lower than those required to block cell adhesion on purified matrix components and are sufficient to occupy less than 10% of the alpha 6 or beta 1 subunits available at the cell surface. These results suggest that integrins may be involved in this potential model of angiogenesis in processes beyond their usual role in cell adhesion. Based on these results, it seems likely that the EA hy 926 cell line will prove to be a useful model for in vitro study of angiogenic processes.

Endothelium specific Weibel-Palade bodies in a continuous human cell line, EA.hy926.

Weibel-Palade bodies are ultrastructurally defined organelles found only in vascular endothelial cells. Because endothelium in corpo is very dispersed, isolation and further characterization of this organelle has been dependent on increasing the number of cells in culture. However, primary isolates of endothelial cells have a limited replication potential and tend to senesce in culture. In this report, EA.hy926, a continuously replicating cell line derived from human endothelium, is shown to contain Weibel-Palade bodies. Electron micrographs demonstrate the ultrastructural characteristics of these tissue-specific organelles and their cytoplasmic distribution in EA.hy926 cells. Von Willebrand factor, which has been shown to exist in Weibel Palade bodies, is demonstrated by immunofluorescence in discrete rod-shaped organelles whose size, shape, and distribution are consistent with that of Weibel-Palade bodies in primary endothelial cell cultures. Rapid release of von Willebrand factor can be induced by calcium ionophore, and large multimeric forms of the protein are found in EA.hy926 cells. These two properties are consistent with the function currently ascribed to Weibel Palade bodies: storage of multimerized von Willebrand factor. Thus ultrastructural, immunologic, and functional data establish the existence of this as yet poorly understood tissue-specific organelle in a continuous, vigorously replicating human cell line.

Stability of the thrombin-thrombomodulin complex on the surface of endothelial cells from human saphenous vein or from the cell line EA.hy 926.

Protein C activation by alpha-thrombin on the surface of endothelial cells depends on an essential membrane-glycoprotein cofactor, thrombomodulin. In the present study we have monitored the activity of thrombin-thrombomodulin complexes on human saphenous-vein endothelial cells (HSVEC) or on the endothelial cell line EA.hy 926. Cell monolayers were exposed for 5 min to 8.5 nM human alpha-thrombin and then washed to remove unbound thrombin. The cells were then incubated at 37 degrees C for 5-180 min. At the end of the respective incubation periods, purified human protein C (120 nM) was added in order to assay the activity of the thrombin-thrombomodulin complexes present on the cell surface. HSVEC pre-exposed to thrombin retained their full capacity to promote protein C activation up to 90 min after free thrombin was removed. This capacity then decreased slowly to reach 56% of control value after 180 min of incubation. Original activity was 3.8 +/- 0.9 pmol of activated protein C formed/min per ml per 10(6) cells (mean +/- S.E.M., n = 5). The capacity of protein C activation of EA.hy 926 cells remained constant for 120 min after free thrombin was removed, then decreased to 76% of control after 180 min. Original activity was 2.0 +/- 0.4 pmol of activated protein C formed/min per ml per 10(6) cells (mean +/- S.E.M., n = 3). Similar results were obtained with cells fixed with 3% paraformaldehyde. However, during the 5-180 min incubation period, non-fixed cells of both types were capable of significantly internalizing fluorescent acetylated low-density lipoprotein. In the experimental protocol used here, an eventual inhibition of thrombin internalization by protein C can be excluded, as protein C is only added at the end of the incubation period. We conclude that there is no evidence of rapid internalization of thrombin-thrombomodulin complexes on HSVEC or the EA.hy 926 cell line, as assessed by the ability of membrane-bound thrombin to activate protein C.

Granulocyte- and granulocyte-macrophage-colony stimulating factors induce human endothelial cells to migrate and proliferate.

Granulocyte-colony stimulating factor (G-CSF) and granulocyte-macrophage-colony stimulating factor (GM-CSF) belong to a family of glycoprotidic growth factors required for the survival, growth and differentiation of haematopoietic precursors and which affect the function of circulating mature cells. They are produced by resting or stimulated stromal cells of the haematopoietic microenvironment (fibroblasts and endothelium) and by immunocompetent cells (T cells and monocytes/macrophages). The action of these CSF molecules was thought to be restricted to cells of haematopoietic origin. Here, we report that G-CSF and GM-CSF influence the migration and proliferation of human endothelial cells suggesting that these molecules may act as regulatory signals outside the haematopoietic system.

The distribution of distinct integrins in focal contacts is determined by the substratum composition.

The distribution of two integrins, the fibronectin receptor and the vitronectin receptor, has been explored in an endothelium-derived cell line plated onto various substrata. On a fibronectin substratum, in the presence of serum, these cells develop focal contacts that contain the fibronectin receptor, whereas the vitronectin receptor is diffusely distributed over the cell surface. Conversely, cells plated onto vitronectin-coated coverslips concentrate only the vitronectin receptor within focal contacts. The accumulation of the vitronectin receptor within focal contacts also occurs when the cells are plated on uncoated coverslips but in the presence of serum. Therefore, we conclude that under normal culture conditions (i.e. in serum-containing media), the vitronectin receptor is the predominant form of integrin involved in substratum adhesion. This conclusion is supported by experiments in which cells were cultured on fibronectin-coated coverslips in the presence of serum. Initially these cells developed focal contacts containing only the fibronectin receptor. Within several hours, however, there was a progressive replacement of focal contacts containing the fibronectin receptor by focal contacts expressing the vitronectin receptor. After approximately 12 h in culture, most cells contained focal contacts expressing only the vitronectin receptor. Focal contacts containing either the fibronectin or vitronectin receptor were both associated with the termini of stress fibres and contained the proteins talin and vinculin. These observations lead us to propose that the cell does not discriminate between these different integrins when assembling the cytoskeletal components at the cytoplasmic face of focal contacts.

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.

Prostacyclin expression by a continuous human cell line derived from vascular endothelium.

Prostacyclin is primarily an endothelial cell product. It contributes to the important role of endothelium in maintaining the fluidity of blood by inhibiting platelet aggregation and by promoting vasodilation. Endothelial cells in culture tend to senesce, and the level of prostacyclin expression decreases. A permanent human cell line, EA.hy 926, derived from a fusion of primary endothelial cells with cells of a less differentiated line, has been found to sustain basal and stimulated levels of prostacyclin synthesis.

Isolation of a human von Willebrand factor cDNA from the hybrid endothelial cell line EA.hy926.

A permanent human hybrid endothelial cell line (EA.hy926) was shown to produce the von Willebrand factor, a protein of 250,000 relative mass (Mr) which was secreted into the medium as a 220,000 Mr protein. A cDNA library was constructed in lambda gt11 using mRNA from these hybrid cells. Several von Willebrand factor cDNA clones were isolated from this library using a synthetic oligodeoxyribonucleotide as a hybridization probe. These cDNA clones were used to analyze the von Willebrand factor gene in normal individuals and in cultured cells.

Characterization of human blood coagulation factor XII cDNA. Prediction of the primary structure of factor XII and the tertiary structure of beta-factor XIIa.

A human liver cDNA library was screened by colony hybridization with two mixtures of synthetic oligodeoxyribonucleotides as probes. These oligonucleotides encoded regions of beta-factor XIIa as predicted from the amino acid sequence. Four positive clones were isolated that contained DNA coding for most of factor XII mRNA. DNA sequence analysis of these overlapping clones showed that they contained DNA coding for part of an amino-terminal extension, the complete amino acid sequence of plasma factor XII, a TGA stop codon, a 3' untranslated region of 150 nucleotides, and a poly(A)+ tail. The cDNA sequence predicts that plasma factor XII consists of 596 amino acid residues. Within the predicted amino acid sequence of factor XII, we have identified three peptide bonds that are cleaved by kallikrein during the formation of beta-factor XIIa. Comparison of the structure of factor XII with other proteins revealed extensive sequence identity with regions of tissue-type plasminogen activator (the epidermal growth factor-like region and the kringle region) and fibronectin (type I and type II homologies). As the type II region of fibronectin contains a collagen-binding site, the homologous region in factor XII may be responsible for the binding of factor XII to collagen. The carboxyl-terminal region of factor XII shares considerable amino acid sequence homology with other serine proteases including trypsin and many clotting factors. A preliminary structural model of beta-factor XIIa is proposed based on the known high resolution x-ray diffraction structures of trypsin, chymotrypsin, and elastase.