A permanent human cell line, EA hy 926, has been established that expresses at least one highly differentiated function of vascular endothelium, factor VII-related antigen. This line was derived by fusing human umbilical vein endothelial cells with the permanent human cell, line A549. Hybrid cells that survived in selective medium had more chromosomes than either progenitor cell type and included, a marker. chromosome from the A549 line. Factor VIII-related antigen can be identified intracellularly in the hybrids by immunofluorescence and accumulates in the culture fluid. Expression of factor VIH-related antigen by these hybrid cells has been maintained for more than 100 cumulative population doublings, including more than 50 passages and three cloning steps. This is evidence that EA-hy 926 represents a permanent line.Differentiated functions of endothelium are critical for the vascularization process in normal and neoplastic tissue, for maintaining the blood-brain barrier, and for hemostasis. Factor VIIIrelated antigen (VIIIR:Ag) is an endothelial cell product (1) involved in the aggregation of platelets, and megakaryocytes are the only other cell type known to express this antigen (2).VIIIR:Ag is present in normal human plasma at about 10 pug/ ml, and decreased levels are found in classical von Willebrand disease, an autosomal dominantly inherited bleeding disorder in humans. VIIIR:Ag circulates as a large molecular complex and its platelet-aggregating activity is directly related to the size distribution of the complex (3). Factor VIII coagulant activity (antihemophilic factor) is also associated with the VIIIR:Ag complex in plasma. We have previously described interspecies hybrids between human endothelial cells and a number of rodent cell lines, in which VIIR:Ag was not expressed (7). There has also been a preliminary report by others (8) of human-rodent hybrids that may express VIIIR:Ag, but the intracellular distribution of antigenicity that they detect is not similar to that of VIIIR:Ag in endothelial cells.Here we report a human intraspecies hybrid, EA-hy 926, that expresses VIIIR:Ag with the same morphological distribution as in primary endothelial cells. MATERIALS AND METHODSCell Culture. Cells were cultured on plastic ware at 370C in a humid atmosphere containing 7% CO2 in air. Culture medium was exchanged every 3-5 days. At confluence, 0.01% trypsin (TRL Worthington, no. LSOO 044 52) in 8 mM phosphate-buffered saline (pH 7.4) with 0.54 mM EDTA and 5.5 mM glucose was used to detach the cells, which were then subcultured at lower cell density.Human umbilical vein endothelial cells (HUV-EC) were isolated as described in detail by Gimbrone (9). The vein of an umbilical cord kept at 40C for 4 hr postpartum was irrigated with phosphate-buffered saline. The endothelial cells were dissociated from the vessel wall with 0.1% collagenase (Sigma, no. C2139) in phosphate-buffered saline at 370C for 20 min. The cells were separated from the collagenase solution by centrifugation and were distributed over...

Testican-1, a secreted proteoglycan enriched in brain, has a single thyropin domain that is highly homologous to domains previously shown to inhibit cysteine proteases. We demonstrate that purified recombinant human testican-1 is a strong competitive inhibitor of the lysosomal cysteine protease, cathepsin L, with a K i of 0.7 nM, but it does not inhibit the structurally related lysosomal cysteine protease cathepsin B. Testican-1 inhibition of cathepsin L is independent of its chondroitin sulfate chains and is effective at both pH 5.5 and 7.2. At neutral pH, testican-1 also stabilizes cathepsin L, slowing pH-induced denaturation and allowing the protease to remain active longer, although the rate of proteolysis is reduced. These data indicate that testican-1 is capable of modulating cathepsin L activity both in intracellular vesicles and in the extracellular milieu.

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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.

SummaryVascular 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 104 different genes are thought to be expressed in the typical mammalian cell, our data indicate that about 103 gene products contribute to the differentiated properties of endothelial cells.

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.

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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.