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Plasma tissue plasminogen activator (tPA) has long been considered to be the product of the endothelial cells that line the various parts of the vascular system regardless of vessel size or location. To determine whether this was truly the case in vivo, the distribution of tPA in the endothelium of the mouse lung and other tissues was evaluated. Immunohistochemical analysis of normal lung tissue showed positive staining limited to the endothelial cells of the bronchial arteries regardless of size with few cells of the pulmonary circulation associated with tPA. The pulmonary vessels that did contain endothelial cell-derived tPA were consistently between 7 and 30 µm in diameter. No capillary or large vessel pulmonary endothelium ever stained positive. These results were also observed in primate lung tissue where the bronchial endothelium of all vessels, even down to capillary size, contained tPA while none of the pulmonary endothelium did. Prolonged exposure of mice to hyperoxic conditions promotes acute lung injury and associated inflammation. Using this model, the effect of inflammation on endothelial cell tPA expression was evaluated. A 4.5-fold increase in the number of pulmonary vessels staining positive for tPA was observed after 66 hours with all of these vessels having a diameter between 7 and 30 µm. Again, none of the endothelium of large arteries or veins nor the capillaries had tPA. Whole tissue tPA mRNA increased dramatically with hyperoxia and in situ hybridization analysis showed tPA mRNA in the endothelium of the same types of vessels as antigen. The tPA localized to both the bronchial and pulmonary endothelium was active with neither tPA-PAI-1 complexes nor urokinase found in perfused lung tissue. These results indicate that endothelial cell tPA expression, either constitutive or induced by a pathologic event, is a function of a highly select group of endothelial cells which are defined by their association with vessels of discrete size and/or anatomic location. Thus, the widely held concept that the steady state level of plasma tPA is maintained through its constitutive production by all endothelial cells of the vascular system is invalid. Also suggested is the possibility that endothelial cell tPA might play a broader role than simply maintaining vessel patency as a component of the fibrinolytic pathway and contribute to complex dynamic processes such as inflammation.

The plasminogen activator inhibitor from human endothelial cells (PAI-1) exists in two forms in the culture medium: an active form that binds to and inactivates plasminogen activators and a latent form that in its native state has no anti-activator activity. Inhibitor activity associated with the latent form can be generated by treatment with protein denaturants and makes up more than 98% of the total inhibitor activity in conditioned medium. Plasminogen activator inhibitor activity is also found in cell cytosol. This inhibitor activity is stable to SDS-treatment but is not enhanced by it. We investigated the relationship between this active cell-associated inhibitor and the latent PAI-1 found in the conditioned medium. Both intracellular and extracellular inhibitors were immunoprecipitated by a monoclonal antibody produced against the latent inhibitor from HT1080 fibrosarcoma cells and electrophoresis on SDS gels of various acrylamide concentrations demonstrated that both forms had the same Mr. Incubation of cytosol inhibitor at 37°C resulted in a decline in inhibitor activity with a half-life of ~4 hours, a rate of decline similar to that of the active PAI-1 in conditioned medium, with less than 10% of the original activity present after eight hours. This decline is accelerated at higher temperatures and is not affected by the presence of a variety of protease inhibitors. Approximately 90% of the activity can be regenerated after SDS treatment suggesting that the cell associated inhibitor, during incubation at 37°C, converts to a form similar to that found in conditioned medium. Despite these similarities, the apparent Stoke's radii of the active intracellular inhibitor and the latent inhibitor in conditioned medium were significantly different with values of 2.77 nm and 2.40 nm for active and latent PAI-1, respectively. Incubation of the active form at 37°C resulted in the shift of the Stoke's radius to that similar to the latent PAI-1 (2.45 nm). Thus, the active and latent PAI-1, while being immunologically similar and of the same apparent Mr, can be differentiated by their behavior on gel permeation columns. This suggests that the intracellular inhibitor is a precursor to the latent form.

Trans retinoic acid (t-RA) stimulated the production of tissue plasminogen activator (tPA) in HeLa-S3 and human umbilical vein endothelial cells (huvecs) in a dose-dependent manner with maximal release (four to five times control) at 40 nmol/L and 40 mumol/L, respectively. In endothelial cells, the stimulation of tPA production by phorbol 12-myristate 13-acetate (PMA) was potentiated 1.9-fold by 10 mumol/L t-RA, or 1.8 times the additive effect. In HeLa cells, total tPA secretion with 10 nmol/L PMA was increased from 43 ng/mL to 96 ng/mL by 40 nmol/L t-RA, which was two times the additive effect. Higher concentrations of t-RA (400 nmol/L) depressed tPA secretion by itself and also suppressed PMA-induced tPA production by 50%. Histamine and thrombin also synergized with t-RA. t-RA (40 nmol/L) and 10 micrograms/mL histamine or 10 U/mL thrombin combined to induce tPA production 3.4 and 1.3 times the additive effect in HeLa cells. Cyclic adenosine monophosphate (cAMP) levels were not significantly affected by 10 nmol/L to 10 mumol/L t-RA. Nor did 10 nmol/L PMA and 40 nmol/L t- RA together affect cAMP levels, suggesting that t-RA-mediated potentiation of PMA-induced tPA production occurred via a mechanism that was independent of cAMP levels. Downregulation of protein kinase C (PKC) by pretreatment of huvecs with 100 nmol/L PMA completely blocked a secondary response to PMA, but did not have a significant effect on t- RA induction. Pretreatment with 10 mumol/L t-RA, on the other hand, did not significantly affect a secondary stimulus by 100 nmol/L PMA, but completely suppressed a secondary stimulation by 10 mumol/L t-RA alone. These studies suggest that the mechanism mediating t-RA stimulation of tPA production interacts with the PKC pathway, resulting in synergism.