Alterations in endothelial cell proteinase and inhibitor polarized secretion following treatment with interleukin-1, phorbol ester, and human melanoma cell conditioned medium.

Polarized secretion of matrix metalloproteinases and plasminogen activators by monkey aortic endothelial cells was studied in vitro, using transwell inserts. The endothelial cells constitutively expressed matrix metalloproteinase-2, tissue inhibitors of metalloproteinases 1 and 2, urokinase, and tissue plasminogen activator, all with basal preference. Matrix metalloproteinase-9 activity was induced by phorbol 12-myristate 13-acetate (apical), interleukin-1 alpha (basal), and by conditioned medium from DX3 human melanoma cells (basal). The DX3 melanoma conditioned medium also stimulated basal secretion of matrix metalloproteinase-2, urokinase, tissue plasminogen activator, and tissue inhibitors of metalloproteinases. The rise in proteolytic activity in the basal direction was reflected by increased capacity to degrade subendothelial basement membrane type IV collagen, shown immunohistologically, using monkey kidney tissue sections and basement membrane deposited by endothelial cells into the transwell membrane. Thus, IL-1 alpha and DX3 melanoma conditioned medium can stimulate endothelial cells in vitro to concentrate secretion of proteinases spatially onto the underlying basement membrane. We suggest that the stimulation of endothelial cell proteinase activity by tumor cells may facilitate tumor cell extravasation.

Immunomagnetic separation as a final purification step of liver endothelial cells.

We describe a fast and reproducible method that can be used as a final step in obtaining pure populations of liver endothelial cells. This method employs endothelial cell specific lectin covalently bound to magnetic polystyrene beads (Dynabeads). Evonymus europaeus agglutinin (EEA)-coated Dynabeads were used to purify monkey liver endothelium from Percoll gradient separated nonparenchymal cells. EEA-coated beads were also successfully used to purify monkey aortic endothelial cells. The endothelial cells grew to confluence as a cobblestonelike monolayer, expressed Factor VIII related antigen, and incorporated acetylated-low density lipoprotein. The magnetic beads seemed not to modify the normal properties of the isolated endothelium, thus facilitating their use in experimental studies. This immunomagnetic separation technique may be applicable for purification of endothelial cells from a wide variety of tissue sources.

Immunomagnetic separation as a final purification step of liver endothelial cells.

We describe a fast and reproducible method that can be used as a final step in obtaining pure populations of liver endothelial cells. This method employs endothelial cell specific lectin covalently bound to magnetic polystyrene beads (Dynabeads). Evonymus europaeus agglutinin (EEA)-coated Dynabeads were used to purify monkey liver endothelium from Percoll gradient separated nonparenchymal cells. EEA-coated beads were also successfully used to purify monkey aortic endothelial cells. The endothelial cells grew to confluence as a cobblestonelike monolayer, expressed Factor VIII related antigen, and incorporated acetylated-low density lipoprotein. The magnetic beads seemed not to modify the normal properties of the isolated endothelium, thus facilitating their use in experimental studies. This immunomagnetic separation technique may be applicable for purification of endothelial cells from a wide variety of tissue sources.

Basement membrane type IV collagen degradation: evidence for the involvement of a proteolytic cascade independent of metalloproteinases.

Current hypotheses suggest that both the plasmin system and metalloproteinases are involved in tumor invasion of basement membrane. In this study, we demonstrate that plasmin can directly degrade native and denatured type IV collagen in solution as well as in tissue sections. Tumor cell lines secreted plasminogen activators into culture supernatants that activated exogenous plasminogen to degrade type IV collagen in zymograms and to remove collagen IV immunoreactivity from tissue sections. Inhibition of metalloproteinase activity in culture supernatants by EDTA did not interfere with plasminogen-mediated type IV collagen degradation. We propose that tumor cells possess a mechanism for the degradation of basement membrane type IV collagen, independent of metalloproteinases but dependent on plasminogen conversion to plasmin.

Prognostic indicators of tumor response to Staphylococcus aureus Cowan strain I plasma perfusion.

Ten tumor-bearing dogs were treated with passage of autologous plasma over fixed Staphylococcus aureus Cowan strain I. Five similar dogs were treated identically except for the exposure to S. aureus. These animals have been assessed to identify positive and negative prognostic variables for response. Nonresponder treated animals had significantly larger chest wall tumor bulk than did the responder and control groups (P less than .01). Responder animals had fewer initial circulating immune complexes than did the nonresponders, though each group had similar reductions in immune complexes with therapy. Nonresponder animals had smaller volumes of plasma processed per kilogram of body weight per procedure than did controls (P = .016), whereas responder and control animals had similar volumes processed per kilogram of body weight per procedure (P = .84). These data suggest that the response observed in our original series was significantly related to the larger amount of plasma treated per procedure and suggest that a factor may be eluted from the S. aureus cartridge that mediates this response.

A modification of the canine carotid-jugular shunt.

A shunt modification was used for implantation in the carotid-jugular vessels of the dog. The shunt was made of plastic tubing and was tied to each vessel with three silk ligatures. The tubing was exteriorized and anchored to the skin. The exposed tubing was cut and attached to the input and output lines of a tubing kit for leukapheresis with a continuous flow centrifuge. Surgical implantation of the shunt was accomplished with ease and minimum trauma to the animal. Useful life of the shunt extended over a 3-week period with minimal requirements for anticoagulants.