An Endothelial Storage Granule for Tissue-Type Plasminogen Activator

In previous studies we have shown that, after stimulation by a receptor ligand such as thrombin, tissue-type plasminogen activator (tPA) and von Willebrand factor (vWf) will be acutely released from human umbilical vein endothelial cells (HUVEC). However, the mechanisms involved in the secretion of these two proteins differ in some respects, suggesting that the two proteins may be stored in different secretory granules

Nanoparticles-A Thoracic Toxicology Perspective

A substantial literature demonstrates that the main ultrafine particles found in ambient urban air are combustion-derived nanoparticles (CDNP) which originate from a number of sources and pose a hazard to the lungs. For CDNP, three properties appear important-surface area, organics and metals. All of these can generate free radicals and so induce oxidative stress and inflammation. Inflammation is a process involved in the diseases exhibited by the individuals susceptible to the effects of PM-development and exacerbations of airways disease and cardiovascular disease. It is therefore possible to implicate CDNP in the common adverse effects of increased PM. The adverse effects of increases in PM on the cardiovascular system are well-documented in the epidemiological literature and, as argued above, these effects are likely to be driven by the combustion-derived NP. The epidemiological findings can be explained in a number of hypotheses regarding the action of NP:-1) Inflammation in the lungs caused by NP causes atheromatous plaque development and destabilization; 2) The inflammation in the lungs causes alteration in the clotting status or fibrinolytic balance favouring thrombogenesis; 3) The NP themselves or metals/organics released by the particles enter the circulation and have direct effects on the endothelium, plaques, the clotting system or the autonomic nervous system/ heart rhythm. Environmental nanoparticles are accidentally produced but they provide a toxicological model for a new class of purposely 'engineered' NP arising from the nanotechnology industry, whose effects are much less understood. Bridging our toxicological knowledge between the environmental nanoparticles and the new engineered nanoparticles is a considerable challenge

Adenosine 3’:5’-Cyclic Monophosphate Induces Regulated Secretion of Tissue-Type Plasminogen Activator and von Willebrand Factor from Cultured Human Endothelial Cells

SummaryThe effect of compounds increasing intracellular adenosine 3’:5’-cyclic monophosphate [cAMP]i levels (prostacyclin, isoproterenol, forskolin, cholera toxin), and of the cAMP analogs 8-bromo-cAMP and dibutyryl-cAMP, on the regulated secretion (acute release) of tissue-type plasminogen activator (tPA) and von Willebrand factor (vWF) was studied in cultured human umbilical vein endothelial cells (HUVEC).Prostacyclin, isoproterenol and forskolin, which increased [cAMP]i in HUVEC, and the cell-permeant cAMP analog 8-bromo-cAMP induced dose- and time-dependent secretion of tPA and vWF. The extent of vWF and tPA release correlated with [cAMP]i, and was increased by the phosphodiesterase inhibitor isobutylmethylxanthine.In contrast to thrombin, the cAMP-elevating agents did not increase the intracellular calcium concentration [Ca2+]i in HUVEC. At sub-maximal concentrations, the effects of thrombin and prostacyclin were additive.Our results show that an increase in [cAMP]i resulted in regulated secretion (acute release) of tPA and vWF from HUVEC, without the concomitant increase in [Ca2+]i which is, in HUVEC, essential for thrombin-induced regulated secretion to occur. cAMP-induced secretion represents a novel mechanism for causing regulated secretion of tPA and vWF from endothelial cells.</jats:p

Studies on the acute release of tissue-type plasminogen activator from human endothelial cells in vitro and in rats in vivo: evidence for a dynamic storage pool

The process of acute release of tissue-type plasminogen activator (tPA) is important in locally speeding up fibrinolysis. Using a sensitive enzyme-linked immunosorbent assay for tPA, we investigated the acute release of tPA from cultured human umbilical vein endothelial cells. The addition of thrombin (0.003 to 3 NIH U/mL) caused the dose-dependent release of noncomplexed, enzymatically active tPA into the medium. The amount of tPA released into the medium by thrombin was similar to the difference in the amounts of tPA present in extracts from thrombin-treated cells and control cells. The process of acute release of tPA was complete in 1 minute, whereas the concomitant release of von Willebrand factor into the medium was slightly slower (maximum after 3 minutes). By increasing (c.q. decreasing) tPA synthesis, it was found that the amount of tPA constitutively secreted, the amount acutely released, and the amount in cell extracts were increased (c.q. decreased) to the same extent. The same relation was found in vivo. When rats were pretreated with cholera toxin or retinoic acid to increase tPA synthesis, plasma levels of tPA were increased, whereas acute release of tPA, as induced by bradykinin, was increased to the same extent. Acutely released tPA and constitutively secreted tPA were liberated from different pathways in human umbilical vein endothelial cells; tPA had, relative to the in vivo situation, a short residence time in the acutely releasable pathway.</jats:p

Endogenous Nitric Oxide Contributes to Bradykinin-Stimulated Glucose Uptake but Attenuates Vascular Tissue-Type Plasminogen Activator Release

Bradykinin causes vasodilation, stimulates tissue-type plasminogen activator (t-PA) release and, in rodents, increases muscle glucose uptake. Although bradykinin causes vasodilation partly by activating nitric-oxide synthase (NOS), the role of nitric oxide in regulating bradykinin-stimulated t-PA release is uncertain. This study examined the effect of high-dose NOS inhibition on bradykinin-stimulated t-PA release and glucose uptake in humans. We studied 24 healthy (12 women and 12 men), overweight and obese (body mass index >25 kg/m2), normotensive, nondiabetic subjects with normal cholesterol. We measured the effect of intra-arterial Nω-monomethyl-l-arginine (l-NMMA, 12 μmol/min) on forearm blood flow (FBF), net t-PA release, and glucose uptake at baseline and in response to intra-arterial bradykinin (50–200 ng/min) in subjects pretreated with the cyclooxygenase inhibitor aspirin. Measurements were repeated after isosorbide dinitrate (ISDN; 5 mg) or sildenafil (50 mg). l-NMMA decreased baseline FBF (P < 0.001), increased baseline forearm vascular resistance (P < 0.001), and increased the t-PA arterial-venous gradient (P = 0.04) without affecting baseline net t-PA release or glucose uptake. During l-NMMA, ISDN tended to decrease baseline net t-PA release (P = 0.06). l-NMMA blunted bradykinin-stimulated vasodilation (P < 0.001 for FBF and FVR). Bradykinin increased net glucose extraction (from −80 ± 23 to −320 ± 97 μg/min/100 ml at 200 ng/min bradykinin, P = 0.02), and l-NMMA (−143 ± 50 μg/min/100 ml at 200 ng/min, P = 0.045) attenuated this effect. In contrast, l-NMMA enhanced bradykinin-stimulated t-PA release (39.9 ± 7.0 ng/min/100 ml versus 30.0 ± 4.2 ng/min/100 ml at 200 ng/min, P = 0.04 for l-NMMA). In gender-stratified analyses, l-NMMA significantly increased bradykinin-stimulated t-PA release in women (F = 6.7, P = 0.02) but not in men. Endogenous NO contributes to bradykinin-stimulated vasodilation and glucose uptake but attenuates the fibrinolytic response to exogenous bradykinin