Lung permeability and hemodynamics during endotoxemia: Effect of aprotinin

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Lung Permeability

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and Hemodynamics during Endotoxemia: Effect of Aprotinin

R. WINN, PH.D., J. GLEISNER, PH.D., R. MAUNDER, M.D., J. HARLAN, M.D., AND J. HILDEBPANT, PH.D. Departments of Surgery, Medicine and Physiology-Biophysics. University Washington 98195; and Virginia Mason Research Center, Seattle,

of Washington, Seattle, Washington 98101

Submitted for publication December 26, 1985 To test the hypothesis that the broad spectrum protease inhibitor, aprotinin, can prevent early pathophysiology of sepsis,we administered endotoxin (0.1-0.75 &kg) by a 30-min infusion to awake goats. Animals were used as their own controls receiving endotoxin with no treatment on one day and treatment with a bolus injection (10 trypsin inhibitory units, TIU, per kg) followed by a 6-hr infusion (5 TIU/kg/ hr) of aprotinin on another. The effect on systemic and pulmonary hemodynamics, lung lymph flow (et,), lymph plasma protein ratio (L/P), and systemic eicosanoid levels were assessed.Q,, quickly reached 28 ml/hr (four times baseline) in both groups then slowly returned toward baseline. L/P ratio of both groups decreased by about 10% then returned to baseline. &and L/P were not different between groups. Likewise+ vascular parameters were not different between groups. Mean pulmonary artery pressure increased approximately 150% to a peak of 58 cm Hz0 in both groups while pulmonary artery wedge pressure doubled from a baseline of 8 cm Hz0 then both groups returned to baseline. Systemic arterial pressure decreased over the 6 hr experimental period by 15 Torr to 70 Torr in both groups. Cardiac output declined from 4.3 to 3 liter/min after the endotoxin, remaining at that level for 2 hr then progressively increased to about 5 liter/min in both groups. We conclude that aprotinin, in doses similar to those reported to give protection from acute lung injury of various origins, fails to modify the early cardiopulmonary pathophysiology of endotoxin. 0 1986 Academic Press, hc.

The adult respiratory distress syndrome (ARDS) is a major pulmonary complication in intensive care unit patients. The incidence of ARDS in the United States was estimated at 150,000 cases in 1972 with a mortality of greater than 50% [ 171. Mortality has not declined from the original high percentage despite current supportive therapy [9, 181. Pathogenesis of the syndrome is unknown but it is associated with several risk factors including sepsis, aspiration of gastric contents, pulmonary contusion, multiple emergency transfusions, multiple major bone fractures, near drowning, pancreatitis, and prolonged hypotension. Pulmonary failure in ARDS is thought to result from damage to the alveolarcapillary barrier, causing increased permeability that is characterized by pulmonary edema rich in proteins and inflammatory cells

changes following endotoxin infusion in sheep [ 1 I]. Activated neutrophils accumulate in the lungs where they may cause increased vascular permeability by secretion of toxic oxygen products, proteases or arachidonic acid metabolites [ 121. A number of studies have shown that neutrophils contain proteases with activity at neutral pH [6, 131. Further evidence suggesting a role for proteases in ARDS was found from studies of bronchoalveolar lavage fluids from patients [ 14, 151. These studies showed that proteolytic activity was elevated in lavage fluids from most of the ARDS patients. The protease inhibitor aprotinin (trade name Trasylol) has been used in both patients and experimental animals in an attempt to ameliorate pulmonary insufficiency following various insults. Clinical studies that have reported some protection using aprotinin have examined pulmonary complications resulting from major bone trauma, shock [ 161, and pancreatitis [22]. These prospective studies

[l, 191. Polymorphonuclear leukocytes (neutrophils) have been implicated in permeability 0022-4804/86

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Copyright 0 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.

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showed a decrease in the severity of injury [ 161 and a decrease in mortality [22] when aprotinin was infused after the insult. The dosage used was approximately 10 trypsin inhibitor units (TIU) per kg [ 161 in one study, and 4 TIU/kg in the other [22]. In animal studies, aprotinin protected animals from shock and improved their chance of survival following superior mesenteric artery occlusion [ 51. Also, the increased pulmonary vascular permeability following experimental pancreatitis was completely blocked by aprotinin (approximately 10 TIU/kg bolus followed by 5 TIU/ kg/hr infusion) [ 10, 211. Change in permeability was also blocked in experimental pancreatitis following granulocyte depletion [2]. Protection seen in these experiments may have been the result of preventing complement activation, blocking some coagulation pathway, or blocking fibrinolysis. Alternatively it may have been the result of blocking neutrophil proteases or by preventing neutrophil sequestration and/or activation. Since endotoxin infusion was shown to be granulocyte dependent [ 111, as was experimental pancreatitis [2], we looked for a similar protection from aprotinin in endotoxemia as was seen in pancreatitis. The studies described in this report investigated a possible protective role for aprotinin in lung injury resulting from infusion of Escherichia coli endotoxin. We infused endotoxin into chronically instrumented unanesthetized goats and measured pulmonary and systemic hemodynamics, thromboxane and prostacyclin release, and changes in lung fluid balance. METHODS

Six goats with chronic lung lymph fistulae were prepared by one of two methods as previously described [20, 241 to measure lung lymph flow (Qr). Briefly, in one procedure the largest efferent duct of the caudal mediastinal node (CMN) was isolated and cannulated, and all other efferent ducts ligated. In the other procedure a pouch was formed in the thoracic duct by ligating on both sides of efferent ducts from the CMN then cannulating the pouch.

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The caudal protion of the CMN was then ligated in two places and sectioned between the ligatures. Care was taken to remove diaphragmatic and esophageal lymphatics that supply non-pulmonary lymph. These preparations have been shown to produce lymph that is 80% pure lung lymph. Catheters were placed in the thoracic aorta and vena cava for pressure (P, and Pv) monitoring and blood sampling. Three to four days after the initial surgery a flow directed thermal dilution catheter was placed in the pulmonary artery via the jugular vein to measure cardiac output (Q) pulmonary artery (Ppa), and pulmonary artery wedge (P,) pressures. Experiments were begun on the day following insertion of the flow directed catheter. Lymph flow was determined as the average flow over 15-min intervals by collecting lymph in tared heparinized tubes. Blood was drawn every 30 min and both lymph and blood samples centrifuged within 2 hr. Lymph and plasma samples were frozen for later determination of their lymph to plasma protein concentration ratio (L/P) by a modified biuret technique [8]. Vascular pressures, Qr and Qt were measured every 15 min until a 2-hr steady baseline had been established. The endotoxin was infused over 30 min with a total dose between 0.1 and 0.75 &kg. Experiments were continued for 6 hr after the start of the endotoxin infusion. Each of the animals served as its own control by receiving endotoxin alone as well as endotoxin with a pretreatment bolus injection of aprotinin (10 TIU/kg) followed by an infusion of aprotinin (5 TIU/kg/hr). Experiments were separated by 48 hr and the order was varied. Two milliliters of arterial blood was drawn into 0.5 ml indomethacin-EDTA solution during the baseline period and at 30, 60, 90, 120,180,240,300, and 360 min after the start of endotoxin. The blood was centrifuged at 3500g for 10 min at 4°C and plasma was then decanted and frozen at -35 “C for later determination of thromboxane AZ (TxA2) and prostacyclin (PGI*) concentrations. These eicosanoids were measured as their stable metabolites TxBz and 6-keto-prostaglandin F1, (6-keto PGF,,).

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The above eicosanoid levels were determined by direct assay of unextracted goat plasma samples using commercially available radioimmunoassay kits (New England Nuclear). Matrix effects of proteins present in unknown goat plasma were determined in eicosanoid free plasma prepared in the following way. Blood was drawn into indomethacin EDTA as decribed above. Pooled normal goat plasma was obtained, then incubated with Norit A charcoal (Amend Drug and Chemical Co., Irvington, N.J.) 50 mg/ml for 2 hr with constant stirring to remove all eicosanoids. Charcoal was removed by centrifugation and the charcoal stripped plasma filtered (0.2 mm filter, Millipore Corp., Bedford, Mass.) and frozen at -35°C. Standard curves run in the eicosanoid-free plasma control were identical to those run in buffer alone (i.e., 95% B/B,, 50% B/B,, and 5% BIB, differed by less than 10% between the two standard curves). Cross reactivity of the TxBz antibody at 50% B/B, was PGE2 < 0.2%, PGA2 < 0.2%, PGF2 < 0.2%, and 6-keto-PGF, < 0.2% (New England Nuclear Technical Bulletin NEK007). Cross reactivity of 6-keto-PGF, antibody at 50% B/B, was PGF2 < 7.8%, PGEr < 3%, PGFz < 2.7%, PGE2 < 2%, PGAr < 0.3%, PGA2 < O.l%, TXB2 < 0. I%, and 13, 14 dihydro-15-keto-PGF, < 0.02% (New England Nuclear Technical Bulletin NEK-008). Statistical differences were tested using paired t test and potential false conclusions due to multiple tests were determined as de-

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scribed elsewhere (23). Significance was assumed for P < 0.05. All data in the text were expressed as mean -t standard deviation. However, for clarity standard error bars were used in the figures and were drawn at hourly intervals. In the treated group, bars were drawn as mean minus SE and in the control group bars were drawn as mean plus SE. RESULTS

The eight variables measured in this study were not significantly affected by the treatment with aprotinin. These variables described lung lymph, pulmonary vascular pressures, the systemic circulation, and circulating eicosanoid levels. Lymph (Fig. 1). Average values of QL for each measurement during the 2-hr baseline and the 6 hr following the start of endotoxin infusion are shown in Fig. 1. Baseline flow on the day that animals were treated was slightly, but not significantly, higher than on the control day (7.8 vs 6.7 ml/hr). QL in both control and treated experiments was typical of the endotoxin response reported by others [3, 231. QL had begun to increase by the time the endotoxin infusion was complete and peaked 15 min later. The control group peaked at 28.4 ml/hr and the treated group at 28.2 ml/hr. QL then slowly returned toward baseline throughout the remainder of the 6 hr followup period. The groups decreased at approximately the same rate and no statistical differ-

FIG. 1. Mean & (A) and L/P(B) for control (squares) and aprotinin pretreated animals (circles). Endotoxin was infused as indicated by the bar.

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averaged 8.3 and 8.4 cm Hz0 for control and treated experiments, respectively. It increased in both groups following the start of the endotoxin infusion. In control experiments average P,., peaked at 16.7 cm Hz0 before decreasing back to baseline by 4 hr after the start of endotoxin infusion. After antiprotease treatment average P, peaked slightly lower at 14.3 cm Hz0 then decreased to approximately 10 cm Hz0 at 4 hr after start of endotoxin and remained at that level for the remainder of the FIG. 2. Pulmonary pressures (P, and PJ measured in experiment. Pw and P,., were not significantly animals receiving endotoxin infusions (as indicated by the different between control and treated groups bar). Control and treatment groups are indicated by circles at any measurement time. and squares, respectively. Systemic circulation (Fig. 3). P, in the treated group started at a slightly lower pressure (84.5 Torr) than control (89.4 Torr) but ence appeared at any measurement period. Baseline values of L/P averaged 0.55 for both the values were not significantly different. P, groups and both showed a slight decrease dur- peaked in both groups at the conclusion of the ing the first hour after the start of the endo- endotoxin infusion (control, 112.7 Tot-r and toxin infusion. At 6 hr, the L/P ratio of both treated, 97.7 Torr) dipped into a level valley groups had increased from the nadir back to for about 90 min before showing a second baseline or slightly above in both groups. lower peak. Finally, P, decreased steadily bePulmonary vascular pressures (Fig. 2). low baseline for the remainder of the experiMean values of baseline Ppa were 22.1 cm Hz0 ment in both groups reaching 70 Torr at 6 hr. for control and 22.4 cm Hz0 for treated ex- The difference between groups was not signifperiments. In both sets of experiments the peak icant at any measurement period throughout Ppa was seen at 30 min after the start of the the experiments. Baseline Qt averaged 4.2 liter/ endotoxin infusion at 58.3 and 56.7 cm HI0 min in both groups, but declined to approxifor control and treated groups, respectively. mately 3 liter/min following endotoxin infusion in both groups and remained at that level Ppa then declined sharply by about 25 cm Hz0 for about 24 hr. It then progressively increased over the next 30 min before again increasing to approximately 5 liter/min at the end of the slightly to a second broader peak. It then decreased toward baseline for the remainder of experiment. Qt was comparable between the experiment in both groups. Baseline P, groups throughout this experiment with no 10 . Awotlnln l 8

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FIG. 3. Pa (A) and Qt (B) measured in goats receiving an infusion of endotoxin (as shown by the bar). Control and treatment groups are indicated by circles and squares, respectively.

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significant difference at any of the time points measured. Eicosanoids (Fig. 4). Averaged TxBz increased sharply in the control group from less than 1 rig/ml to a peak of 5.7 rig/ml at the conclusion of the infusion. TxBz in the treated animals increased from less than 1 rig/ml to a peak of 3.3 rig/ml 1 hr after the start of the infusion. These differences were not significant. After the peak, TxB2 concentrations decreased rapidly for approximately 1 hr then more slowly back to near baseline at 6 hr. Averaged 6-keto-PGF,, increased more slowly, reaching a peak at 2 hr after the start of endotoxin infusion. It then declined toward baseline for the next 4 hr. Again, there was no difference between the control and treated groups. Total peripheral white blood cell counts decreased rapidly after endotoxin reaching a minimum at 1 hr then returned slowly back to baseline (data not shown). This decrease was seen in control as well as treated animals and the reduction was not significantly different between the groups. There was a general trend in the treated group toward higher counts at each measurement period following endotoxin except at 360 min. DISCUSSION

The study of experimental pancreatitis by Tahamont et al. [21] and Garcia-Szabo and Malik [lo] provided evidence that low-dose

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aprotinin could significantly alter lung injury due to proteases. Garcia-Szabo and Malik showed that aprotinin prevented the drop in systemic granulocyte count and in fibrinogen concentration after induction of experimental pancreatitis. It is possible that aprotinin provided protection from the vascular injury by preventing these changes. The success of aprotinin in preventing these changes seems remarkable in light of the high levels of endogenous antiprotease present in vivo. The amount of aprotinin given in these experiments should not have significantly raised total antiprotease activity. Nevertheless, since lung injury resulting from experimental pancreatitis was blocked by the protease inhibitor, we hypothesized that other forms of neutrophil dependent lung injury might also be blocked by aprotinin. Changes in lung fluid balance as measured by lymph flow and L/P ratio in both experimental groups were similar to those seen in previous work using endotoxin infusions in this model [3,23]. Brigham et al. [3] have designated the early changes as the hydrostatic phase (phase I) since L/P decreases with increased QL, a characteristic of increased hydrostatic pressure [7]. The second phase (phase II), occurring 4 to 6 hr after endotoxin, was designated the permeability phase since QL was elevated without a decrease in the L/P ratio. Changes in Qr, L/P, and the cardiovascular parameters (P,,, P,.,,P,, and QJ of this study were consistent with the phase I and phase II

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FIG. 4. TX& (A) and 6-keto-F’GF1 (B) for control (squares) and treatment (circles) groups. Endotoxin was infused as shown by the bar.

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description of the pathophysiology of endotoxin infusion. In these experiments, aprotinin had no effect on the measured cardiovascular and lung fluid balance parameters. These studies do not, however, rule out the involvement of proteases in endotoxin-induced pulmonary injury since the concentration of endogenous antiproteases plus aprotinin may have been too small to provide protection in the local region where injury occurred (see below). Nonetheless, the dose was chosen based on values in the literature that had been shown to provide at least some protection from lung injury. It may be concluded that the pathogenesis of pulmonary insufficiency following major bone trauma [ 161 and pancreatitis [ 10, 2 1, 221 is different from changes in lung fluid balance and hemodynamics following endotoxin infusions. Aprotinin is thought to function as an antiprotease thus preventing either the cleavage of benign molecules to their toxic fragments or by preventing direct damage to vascular surfaces by proteases released from granulocytes. Neutrophil proteases cannot be ruled out as causing the change in permeability following endotoxin infusion even though there are large quantities of circulating antiproteases. That is, the stimulated neutrophils can sequester in the lungs adhering to endothelial cells thus producing a local protected environment between the neutrophil and endothelial cell. Concentration of potentially injurious agents released by the neutrophils into this local region can thus overcome the protection of circulating antiproteases to produce a local vascular injury. This could occur by overcoming antiproteases directly or by neutrophil release of toxic oxygen products that inactivate endogenous antiproteases [4]. Local release of oxidants and proteases may interact to allow proteolysis to occur. ACKNOWLEDGMENTS This work was supported, in part, by Research Grants GM 29853 and GM 24990, and Biomedical Research Support Grant RR 05588 from the National Institutes of Health.

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