title: Tumor necrosis factor induction of endothelial cell urokinase-type plasminogen activator mediated proteolysis of extracellular matrix and its antagonism by gamma-interferon.
abstract: Tumor necrosis factor (TNF) has a profound capacity to alter the endothelial cell phenotype that includes morphologic and functional changes that may be central for proinflammatory processes. Recent observations have indicated that TNF can promote the synthesis and secretion of urokinase plasminogen activator (uPA) in low passage human endothelial cells that normally release little uPA. In this report we have confirmed and expanded upon these initial observations in human endothelial cells and describe the ability of gamma-interferon (gamma-IFN) to inhibit TNF-induced uPA synthesis and secretion in a dose-dependent manner (0.025 to 25 ng/mL). Analysis of cell-free conditioned medium derived from gamma-IFN-treated cultures by micro-enzyme-linked immunosorbent assay (ELISA) methodologies using uPA- and plasminogen activator inhibitor type 1 (PAI-1)-specific monoclonal antibodies (MoAbs) indicate that the decrease in uPA activity observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) zymography is a direct result of a decrease in extracellular uPA antigen and is not a consequence of increased PAI-1 antigen. These findings are supported by Northern blot analyses that indicate that gamma-IFN treatment of endothelial cells resulted in a decreased steady state level of uPA messenger RNA (mRNA) with no measurable change in PAI-1 mRNA. This inhibitory response is specific for gamma-IFN because alpha-IFN fails to elicit a similar inhibitory response. In addition, TNF augmented extracellular proteolysis of radiolabeled subendothelial extracellular matrix (ECM) in a dose-dependent manner. The observed increase in ECM degradation mediated by TNF treatment of endothelial cells was dependent on the presence of plasminogen and could be inhibited by an anticatalytic uPA MoAb implying the requirement of proteolytically active uPA in this process. gamma-IFN (25 ng/mL) treatment of endothelial cells antagonized TNF-promoted degradation of radiolabeled ECM at a concentration that completely inhibited TNF-mediated uPA expression and activity. In addition, endothelial cells treated with TNF (18 hours) displayed the ability to invade ECM and reorganize individual cells into tube-like structures that were not evident in untreated control cultures when grown on Matrigel-coated culture dishes. gamma-IFN treatment of endothelial cells propagated on Matrigel was observed to inhibit TNF-mediated ECM invasion and tube formation at concentrations that were analogous to those required for the inhibition of uPA expression and activity. In summary, these observations suggest a novel homeostatic control mechanism for endothelial cell regulation of subendothelial ECM degradation promoted by TNF and inhibited by gamma-IFN.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Institute of Arthritis and Autoimmunity, Miles Research Center, Miles Inc, West Haven, CT. 06516.
authors.forename: M J
authors.initials: MJ
authors.lastname: Niedbala
authors.forename: M S
authors.initials: MS
authors.lastname: Picarella
mesh_terms: D001483:Base Sequence; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000201:enzymology*; D004789:Enzyme Activation; D005109:Extracellular Matrix / Q000378:metabolism / Q000648:ultrastructure; D016326:Extracellular Matrix Proteins / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D066298:In Vitro Techniques; D016898:Interferon-alpha / Q000494:pharmacology; D007371:Interferon-gamma / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D015849:Plasminogen Inactivators / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism*
chemical_list: D016326:Extracellular Matrix Proteins; D016898:Interferon-alpha; D009841:Oligonucleotides; D015849:Plasminogen Inactivators; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D007371:Interferon-gamma; D014568:Urokinase-Type Plasminogen Activator