Modulation of vascular endothelial permeability by thrombin.
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Actin Cytoskeleton↗
Biomedical subjects
F L Minnear
Publications and source records attributed to F L Minnear.
Simultaneous measurement of fluid and protein permeability in isolated rabbit lungs during edema.
Fluid conductance and protein permeability have been studied in isolated perfused lung models of pulmonary edema. However, previous studies have not investigated changes of both fluid conductance and protein permeability in the same isolated lung preparation after injury. Arachidonic acid (AA) metabolites are involved in the inflammatory processes that lead to the development of pulmonary edema. The hemodynamic effects of AA have been well established; however, controversy exists concerning the ability of AA to alter the permeability of the pulmonary microvasculature to fluid and protein. The purpose of this study was to simultaneously determine whether transvascular fluid conductance and protein permeability are increased in isolated perfused rabbit lungs with pulmonary edema induced by AA. Indomethacin (80 microM) was added to the perfusate to inhibit the hemodynamic effects of AA and produce a pressure-independent model of pulmonary edema. Fluid conductance was assessed by determination of the capillary filtration coefficient (Kf), and protein permeability was evaluated by measurement of 125I-albumin clearance. The injection of AA (3 mg/200 ml of perfusate) into the pulmonary arterial catheter resulted in an increase in lung weight over the remaining 30-min experimental period. Kf (microliter.s-1 x cmH2O-1 x g dry lung-1) was increased (P < 0.05) in AA-treated lungs at 10 and 30 min post-AA injection when compared with control lungs and baseline values (determined 10 min before AA injection). Albumin clearance was also greater (P < 0.05) in lungs that received AA. 125I-albumin clearance was measured at different rates of fluid flux produced by elevation of venous pressure.(ABSTRACT TRUNCATED AT 250 WORDS)
Albumins↗
Thrombin's enzymatic activity increases permeability of endothelial cell monolayers.
Human alpha-thrombin increases the permeability of bovine pulmonary artery endothelial cell (CCL-209) monolayers. To determine if this increase is via an enzymatic or receptor-mediated mechanism, enzymatically active forms of alpha-thrombin and enzymatically inactive forms with cell binding activity were incubated with the monolayers. Enzymatic forms included alpha-thrombin and two digestion products, zeta-thrombin (chymotryptic product with 89% clotting activity) and gamma-thrombin (tryptic product). Enzymatically inactive forms included D-Phe-Pro-Arg-chloromethylketone-(PPACK) alpha-thrombin and diisopropylphosphorofluoridate-(DIP) alpha-thrombin. Cell binding activity of alpha- and PPACK-alpha-thrombin was demonstrated to be similar to each other and comparable to that cited in the literature for DIP-alpha-thrombin. gamma-Thrombin, on the other hand, did not compete for binding of 125I-labeled alpha-thrombin. All enzymatic forms of alpha-thrombin increased endothelial permeability as assessed by the clearance of 125I-albumin across the monolayers. Coincubation of PPACK, an enzymatic site inhibitor, with alpha- or gamma-thrombin prevented the increase in permeability, further indicating that alpha-thrombin increased permeability by its enzymatic activity. Both enzymatically inactive forms of alpha-thrombin with high-affinity binding activity had no effect on permeability. To further examine whether cell binding activity of alpha-thrombin contributed to the increased permeability, a sulfated COOH-terminal fragment of hirudin (hirugen) that binds to the anion-binding site of alpha-thrombin but, unlike hirudin, does not interact with the catalytic site was coincubated with alpha-thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Chloromethyl Ketones↗
Rat platelets adhere to human thrombin-treated rat lungs under flow conditions.
Platelet attachment to thrombin-treated endothelium was examined under flow conditions in the vascular bed of isolated perfused rat lungs. The infused thrombin bound to the pulmonary endothelium in a specific, competitive manner as shown by displacement of 125I-alpha-thrombin with an excess of cold diisopropylfluoro-phosphate-alpha-thrombin. alpha-Thrombin significantly increased the attachment of isolated 51Cr-labelled rat platelets to the perfused lungs. Scanning and high voltage electron microscopy showed single platelets in various stages of activation attached to pulmonary endothelium. The receptor binding site and catalytic activity of thrombin were essential to the enhancement of platelet attachment. Aspirin given to rats before the lung isolation had no effect on thrombin-induced platelet attachment. Thus, endothelial bound thrombin initiates platelet activation and enhances subsequent platelet attachment.
Animals↗
Platelets decrease albumin permeability of pulmonary artery endothelial cell monolayers.
Since platelets may modulate endothelial cell permeability, we examined the effects of platelets on 125I-albumin permeability of cultured bovine pulmonary artery endothelial cell monolayers. The experimental system consisted of endothelial cells grown to confluence on a gelatinized polycarbonate filter. We quantified the diffusive flux of 125I-albumin from luminal chamber to the abluminal chamber. Washed human platelets added to the monolayers decreased the albumin flux in a concentration-dependent manner, with a 65% decrease occurring at the highest concentration of platelets (5 x 10(7) platelets) added to the 700-microliters luminal chamber. In contrast, neither paraformaldehyde-fixed platelets nor fresh red blood cells changed 125I-albumin permeability. Platelets had no effect on 125I-albumin permeability across the gelatinized filters without endothelial cells present. Supernatants of platelet lysates also reduced albumin flux. The effect produced by intact platelets or platelet lysate was not influenced by the presence of ketanserin (a serotonin receptor antagonist), propranolol (a beta-adrenergic receptor antagonist), or aspirin (an inhibitor of cyclooxygenase). Platelets activated by thrombin did not produce an effect that was different from the effect produced by intact platelets. The activity of the supernatant of platelet lysate remained in the aqueous phase after ether extraction. The results indicate that the platelet-mediated decrease in endothelial cell permeability to 125I-albumin is the result of a hydrophilic platelet-derived factor(s) and not secondary to mechanical obstruction of endothelial "leaks" by the platelets.
Albumins↗
Platelets adhere to thrombin-treated endothelial cells in vitro.
Interaction of thrombin with vascular endothelial cells was investigated as a mechanism promoting platelet activation and adherence to endothelial monolayers. We found that pretreatment of endothelium with alpha-thrombin in the absence of platelets results in the attachment of platelets to endothelial cells after the removal of fluid-phase alpha-thrombin. This activity was eliminated by exposure of alpha-thrombin-pretreated endothelial cells to active site inhibitors of alpha-thrombin or by adding alpha-thrombin in the presence of excess diisopropyl fluorophosphate-inhibited thrombin, suggesting retention of active alpha-thrombin by a receptor-mediated mechanism. Morphological data and the results of [14C]serotonin release studies indicate that platelets are activated by alpha-thrombin-pretreated endothelium and that adherence represents aggregates of activated platelets as well as individual platelets. Adherence on alpha-thrombin-pretreated endothelium is dependent on divalent cations. Platelets also adhered to aortic segments pretreated with thrombin. The data of the current studies support the contention that alpha-thrombin can promote adherence of activated platelets to endothelial cells because of the binding and retention of alpha-thrombin to endothelial cells in a manner in which it remains active and available for platelet activation.
Animals↗
Isoproterenol reduces thrombin-induced pulmonary endothelial permeability in vitro.
The ability of the beta-adrenergic agonist, isoproterenol, to attenuate the thrombin-induced increase in endothelial permeability was examined by measuring 125I-labeled albumin clearance across endothelial cell monolayers. Bovine pulmonary artery endothelial cells (CCL-209) were grown to confluence on gelatinized, polycarbonate micropore filters and mounted on modified Boyden chambers with Dulbecco's modified Eagle's medium (DMEM) and 0.5% bovine serum albumin. alpha-Thrombin at 0.2 nM to 2 microM produced a dose-related increase (P less than 0.01) in 125I-labeled albumin clearance from the DMEM control value. Light and electron microscopy revealed that the thrombin-induced increase in permeability correlated with changes in cell shape and rearrangement of filamentous actin. Coincubation of 2 microM isoproterenol with 2 microM alpha-thrombin reduced (P less than 0.01) the thrombin-induced increase in albumin clearance and the observed morphological changes. This attenuation was not caused by inhibition of thrombin's enzymatically active site, since isoproterenol did not impair thrombin's fibrinogen clotting activity nor its amidolytic cleavage of an artificial substrate (Spectrozyme-TH). Coincubation of 20 microM propranolol, a beta-adrenergic antagonist, with 2 microM isoproterenol and thrombin blocked the permeability-decreasing effect of isoproterenol. Both 2 microM isoproterenol and 2 pM alpha-thrombin alone decreased (P less than 0.01) albumin clearance below the DMEM control value. These results suggest that isoproterenol can reduce the thrombin-induced increase in endothelial permeability in vitro by directly maintaining actin filaments and the shape of endothelial cells.
Animals↗
Effect of ibuprofen and dexamethasone on Kupffer cell complement receptor function after endotoxemia and the phagocytosis of erythrocytes.
Depression of Kupffer cell complement receptor (CR) function is associated with several states of depressed host defense. This study was carried out to determine if ibuprofen and dexamethasone, which decrease the mortality rate following endotoxemia, could prevent the depression of CR function caused by endotoxemia and the phagocytosis of antibody-coated erythrocytes (EIgG). The depression of CR function caused by endotoxin was completely prevented by the administration of ibuprofen or dexamethasone. Thus, the ability of these drugs to prevent the depression of macrophage function may contribute to their salutory effects during endotoxin shock. In contrast to the effect with endotoxemia, the depression of CR function caused by the phagocytosis of EIgG was not modified by pretreatment with ibuprofen or dexamethasone. Additional studies demonstrated that the depression of CR function caused by EIgG was probably not due to EIgG in the blood or bound to Kupffer cells, interfering with the receptor probe for access to the CR. This study has shown that ibuprofen and dexamethasone can prevent the depression of CR function caused by endotoxin but not the depression caused by the phagocytosis of EIgG. These results suggest that different mechanisms mediate the depression of CR function caused by endotoxin and the phagocytosis of EIgG.
Animals↗
Effect of Kupffer cell phagocytosis of erythrocytes and erythrocyte ghosts on susceptibility to endotoxemia and bacteremia.
The phagocytosis of erythrocytes by macrophages has previously been shown to depress macrophage function. In this study we compared the effect of the phagocytosis of erythrocytes and erythrocyte ghosts by Kupffer cells on the duration of the depression of complement receptor clearance function and host defense against endotoxemia and bacteremia. Phagocytosis of erythrocytes and erythrocyte ghosts was induced in rats by the injection of rat erythrocytes or erythrocyte ghosts coated with anti-rat erythrocyte immunoglobulin G (EIgG and GIgG, respectively). The hepatic uptake of EIgG and GIgG (17.4 X 10(8)/100 g) occurred during the first 30 min after injection. The digestion of phagocytized EIgG and GIgG, as assessed by electron microscopy, was complete at 24 and 3 h after injection, respectively. The depression of Kupffer cell complement receptor clearance function caused by EIgG and GIgG returned to normal by 6 h after injection of EIgG and by 3 h after injection of GIgG. Phagocytosis of EIgG depressed the survival rate after endotoxemia and bacteremia when endotoxin or bacteria were injected at 30 min after EIgG. The survival rate returned to normal when the endotoxin and bacteria were injected at 12 and 6 h after the EIgG, respectively. Phagocytosis of GIgG did not depress the survival rate after endotoxemia and bacteremia. Thus, compared with erythrocytes, erythrocyte ghosts are more rapidly digested after phagocytosis, depress complement receptor function for a shorter period of time, and cause less depression of host defense. These findings indicate that the contents of erythrocytes play an important role in the impairment of host defense caused by the phagocytosis of erythrocytes by Kupffer cells.
Animals↗
Lung morphological and permeability changes induced by intravascular coagulation in dogs.
The objectives of this study were to describe the morphological alterations of the pulmonary microvasculature in anesthetized dogs infused intravenously with alpha-thrombin (111-233 NIH U/kg) and to measure the protein reflection coefficient (sigma d), an index of pulmonary vascular permeability to proteins. Quantitative electron microscopy of lung biopsies from a group of four dogs showed an increase in the volume density of the alveolar interstitium and an increase in intravascular fibrin at 30 min after the thrombin infusion. The pulmonary microvascular endothelium was often swollen, blistered, highly vacuolized, or rarefied when close to or in contact with fibrin but appeared normal when not closely associated with fibrin. Fibrin-endothelial interactions often included projections of endothelium surrounding fibrin, the presence of fibrin within endothelial cells, 10- to 15-nm bridges connecting fibrin and endothelium, and disruption of the endothelial plasmalemma in contact with fibrin. Alveolar epithelial cells were unaffected by the thrombin infusion. Pulmonary lymph was collected from an afferent lymphatic to the left tracheobronchial lymph node in a second group of five dogs anesthetized with pentobarbital sodium intravenously. Thrombin increased pulmonary lymph flow by sevenfold and also increased the extravascular lung water-to-bloodless dry lung weight ratio. Left atrial pressure was elevated after thrombin infusion until a filtration-independent state was approached for the calculation of sigma d. Thrombin decreased sigma d from a normal value of 0.62 +/- 0.02 to 0.53 +/- 0.02. Thus thrombin-induced intravascular coagulation in dogs produced focal disruptions of the microvascular endothelium associated with intravascular fibrin and increased the pulmonary vascular permeability to proteins.
Animals↗
Measurement of albumin permeability across endothelial monolayers in vitro.
We have developed an experimental system to measure the permeability of the cultured endothelial monolayer. The luminal-to-abluminal flux of 125I-albumin across cultured pulmonary endothelium was expressed as a clearance rate equal to the permeability-surface area product. After clearance rate measurement for a 30-min base-line period, a test agent was added to the luminal side, and the clearance rate was remeasured during a 30-min experimental period. In control studies the base-line clearance rate was 0.343 +/- 0.017 microliter/min. After correction for the diffusional resistances of the filter and unstirred layers, the calculated permeability of the endothelial monolayer was 1.2 X 10(-5) cm/s. When culture medium was the test agent, the experimental clearance rate was unchanged from the base-line value. After addition of 4 mM oleic acid to the luminal chamber, the clearance rate was 0.528 +/- 0.017 microliter/min compared with a base-line value of 0.330 +/- 0.008 microliter/min (P less than 0.005). This method allows the calculation of endothelial permeability with correction for unstirred layers and the use of each monolayer as its own control.
Animals↗
Endothelial injury and pulmonary congestion characterize neurogenic pulmonary edema in rabbits.
The objectives of the present study were to determine whether an intracisternal injection of fibrinogen-sodium citrate, a model of neurogenic pulmonary edema (NPE), produces protein-rich or protein-poor pulmonary edema, and to determine whether the edema is associated with pulmonary vascular hypertension and pulmonary congestion. Fibrinogen (6-10 mg/ml) dissolved in 0.055 M sodium citrate was injected into the cisterna magna of six New Zealand White rabbits. Six additional rabbits were injected with saline to control for the effects of intracranial hypertension and pulmonary vascular hypertension. The fibrinogen-sodium citrate solution or sodium citrate alone, as opposed to saline, produced systemic and pulmonary vascular hypertension, pulmonary edema, hypoxemia, hypercapnia, and acidosis. The lungs from fibrinogen-injected rabbits were edematous, congested, and liverlike in appearance. Tracheal froth that was blood tinged and protein rich was present in five of the six rabbits. Microscopic examination of lung biopsies revealed erythrocytes and plasma in the alveoli and focal injury to the pulmonary microvascular endothelium. Fibrinogen-sodium citrate increased (P less than 0.05) the extravascular lung water (EVLW) (10.3 +/- 2.0 vs. 5.5 +/- 0.6 g, means +/- SE), lung blood weight (9.7 +/- 1.3 vs. 3.8 +/- 0.6 g), total dry lung weight (3.2 +/- 0.4 vs. 2.0 +/- 0.1 g), and the EVLW-to-blood-free dry lung weight ratio (7.0 +/- 0.8 vs. 4.0 +/- 0.3 g) from saline-control values. There was no difference in the blood-fre dry lung weight (1.4 +/- 0.1 vs. 1.3 +/- 0.1 g) between the two groups. These findings demonstrate that pulmonary congestion, pulmonary vascular hypertension, and focal endothelial injury contribute to the development of NPE.
Animals↗
Beta-adrenergic modulation of pulmonary transvascular fluid and protein exchange.
We determined in anesthetized sheep whether isoproterenol, a beta-adrenergic agonist, prevents the increases in pulmonary fluid and protein exchange produced by thrombin-induced intravascular coagulation. Seven sheep were infused intravenously with 0.05 micrograms X kg-1 X min-1 isoproterenol before infusion of alpha-thrombin, and six sheep were infused with alpha-thrombin only and served as control subjects. The marked increases in pulmonary lymph flow and lymph protein clearance in the control thrombin group were attenuated (P less than 0.05) in the isoproterenol group in association with a higher pulmonary blood flow (P less than 0.05) and a lower pulmonary vascular resistance (P less than 0.05) in the isoproterenol group and with similar increases in pulmonary arterial and pulmonary arterial wedge pressures in both groups. The decreases in fluid and protein fluxes produced by isoproterenol are related to its beta-adrenergic properties because propranolol, a beta-adrenergic antagonist, blocked the protective effects of isoproterenol in a second group of sheep infused with propranolol, isoproterenol, and thrombin. Raising left atrial pressure to test for changes in vascular permeability increased protein flux to a much greater extent in the thrombin control group than in the isoproterenol group challenged with thrombin. The data suggest that isoproterenol attenuated the increase in fluid and protein fluxes produced by thrombin-induced intravascular coagulation by a permeability-decreasing mechanism.
Adrenergic beta-Agonists↗
Hyperthermia-induced pulmonary edema.
The effects of temperature (37-45 degrees C) on pulmonary edema formation and transendothelial albumin clearance were investigated using isolated perfused guinea pig lungs and bovine pulmonary arterial endothelial cells grown to confluency on a gelatinized membrane. Perfusion of isolated lungs with Ringer-albumin solution at 37 or 41 degrees C for 90 min produced no change in lung wet-to-dry weight ratios (W/D) or in pulmonary capillary pressure (measured by the double-occlusion method). When perfused at 43 degrees C, lung wet weight increased 0.8 +/- 0.4 g over base line (final W/D = 7.43 +/- 0.7) within 90 min. Perfusion at 45 degrees C increased lung weight by 2.7 +/- 0.9 g over base line (final W/D 11.8 +/- 2.3 vs. control value of 5.2 +/- 0.23 at 37 degrees C perfusion) within 60 min. The changes in pulmonary capillary pressure were small (from a base-line value of 4.3 +/- 0.8 to 4.9 +/- 0.4 at 43 degrees C and from a base-line value of 4.9 +/- 0.8 to 5.9 +/- 0.6 at 45 degrees C). The clearance of 125I-albumin (microliter/min) across the endothelial monolayer system increased threefold (from 0.295 +/- 0.035 to 1.048 +/- 0.107) at 45 degrees C, an effect comparable to positive controls of trypsin (from 0.272 +/- 0.046 to 1.595 +/- 0.138) or oleic acid (from 0.278 +/- 0.043 to 0.672 +/- 0.26). An increase in temperature from 37 to 45 degrees C had no effect on the permeability of the gelatinized membrane alone.(ABSTRACT TRUNCATED AT 250 WORDS)
Albumins↗