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Anesth Analg 2005;100:929-936
© 2005 International Anesthesia Research Society
doi: 10.1213/01.ANE.0000147707.49192.88

CARDIOVASCULAR ANESTHESIA

Propofol Attenuates Lung Endothelial Injury Induced by Ischemia-Reperfusion and Oxidative Stress

Irina V. Balyasnikova, PhD, David J. Visintine, BA, Helena B. Gunnerson, MD, Chanannait Paisansathan, MD, Verna L. Baughman, MD, Richard D. Minshall, PhD, and Sergei M. Danilov, MD, PhD

Departments of Anesthesiology and Pharmacology, University of Illinois at Chicago, Chicago, Illinois

Address correspondence and reprint requests to Sergei M. Danilov, MD, PhD, Anesthesiology Research Center, University of Illinois at Chicago, 1819 W. Polk St. (M/C 519), Chicago, IL 60612. Address e-mail to danilov{at}uic.edu.


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Lung dysfunction after cardiopulmonary bypass and lung transplantation results from oxidant-mediated cellular damage. Previously, we observed the shedding of angiotensin-converting enzyme (ACE) from the endothelial cell surface to be a more sensitive and earlier marker of oxidative lung endothelial injury than lung wet-to-dry weight ratio. The aim of this study was to evaluate the potential of the anesthetic propofol, which has antioxidant properties, to prevent oxidative lung injury by measuring ACE shedding. ACE release from isolated perfused rat lungs increased significantly after ischemia-reperfusion (I/R). Propofol significantly decreased I/R-induced ACE release by 23.4% (P < 0.05). Perfusion with 0.75 mM H2O2 also caused ACE release from the lung microvasculature, which was similarly attenuated by propofol. The protective effect of propofol on H2O2-induced ACE shedding was confirmed in vitro using Chinese Hamster Ovary cells overexpressing human ACE. Thus, propofol can attenuate oxidative injury of the pulmonary endothelium as detected by ACE shedding in I/R and H2O2 models of acute lung injury.


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Oxidative lung injury occurs during lung transplantation, after cardiopulmonary bypass (CPB), and after lung resection. Lung ischemia induces the generation of oxidants by the pulmonary endothelium leading to oxidative damage to the endothelium (1). Anesthetics could either be protective or exacerbate oxidative damage during lung injury, thus raising important clinical and scientific questions as to the choice of anesthetic in this situation. The commercial formulation of propofol, a lipid-soluble anesthetic, contains {gamma}-tocoferol and {delta}-tocoferol and thus exhibits significant antioxidant activity. Propofol scavenges free radicals, reduces disulfide bonds in proteins, and inhibits lipid peroxidation. Experimental evidence suggests propofol may protect the myocardium and the lung against peroxide (2–5). The volatile anesthetic halothane produced pulmonary microvascular injury (6). On the contrary, other volatile anesthetics such as isoflurane and sevoflurane are thought to be beneficial in cardiac and lung ischemia-reperfusion (I/R) animal models and during CBP (7–11).

Previously, we found that angiotensin-converting enzyme (ACE) shedding from the vascular endothelium in isolated perfused lungs (IPLs) is a more sensitive and earlier marker of oxidative lung endothelial injury than lung wet-to-dry weight ratio (12–13). Therefore, in the present study we have used this cell-specific marker of lung injury to assess the protective effect of propofol on I/R-induced and H2O2-induced lung injury in the rat IPL and on oxidative injury to cells in culture that overexpress ACE.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
All procedures were approved by the University of Illinois Institutional Animal Care and Use Committee. Anesthetic induction of male Sprague-Dawley rats (170–200 g) was accomplished by placing the animal in an enclosed chamber containing isoflurane. After that, the snout of the animal was placed in a tube (sealed about the neck area) connected to the outflow of an anesthetic vaporizer to yield proper anesthetic dose (2.5% for isoflurane) in oxygen. After dissection and en bloc removal of the lung and heart from the thoracic cavity, the lung was mounted in a water-jacketed perfusion chamber maintained at 37°C and allowed to equilibrate for 5 min. During this time, the perfusate was discarded. The perfusate (50 mL per lung of Krebs-Ringer buffer containing 3% bovine serum albumin [BSA]; pH, 7.4) was then re-circulated at 37°C. After an additional 5 min equilibration period, a control perfusate sample was taken and the test anesthetic was introduced for 1 h. I/R injury was induced by stopping the perfusion pump and ventilator for 30 min and then restoring perfusion and oxygenation for 30 min. Perfusate samples were taken every 15 min. For H2O2-induced injury, the rat lung was continuously perfused with re-circulated Krebs buffer containing 0.35 to 1.0 mM H2O2 for 2 h. Perfusion pressure (PP), shedding of ACE from the lung vasculature into the perfusate, and lung wet-to-dry weight ratio were measured in each rat IPL preparation.

Propofol (Diprivan, 2,6 diisopropylphenol) or intralipid vehicle (control) was added to the perfusate to study the effects on lung microvasculature injury. Propofol, dissolved in a lipid emulsion, and vehicle control lipid emulsion were obtained from Novation, LLC and Baxter Healthcare Corp., Deerfield, IL, respectively. Propofol was used at 1x (6.25 µg/mL) and 10x concentrations (62.5 µg/mL). Similar concentrations (5 µg/mL and 50 µg/mL) were used previously (14,15), corresponding to 1x and 10x clinically relevant plasma concentrations based on an study by Shafer (16) stating that the 50 µg/mL concentration would be the maximum dose used clinically for sedation (16). Further reasons to use a wide range of propofol concentrations come from observations that i) propofol solutions (10–100 µM; i.e., our range of concentrations) added to a reservoir consisting of hard glass bottles and plastic tubing lost 80% of their initial concentration in 4 h (17); ii) propofol undergoes extensive uptake and first-pass elimination during recirculation through the lung; 30% of the propofol dose was eliminated during the first pass through the lungs (18). Propofol concentration during the perfusion period was measured by high performance liquid chromatography (HPLC) (18) in the Research Resources Center of the University of Illinois at Chicago. By HPLC determination, more than 90% of propofol (starting dose of 6 µg/mL in the perfusion solution) was no longer detected in the perfusion solution after 30 min. At 1 µg/mL, the depletion of propofol was even more significant (95%). Therefore, the use of propofol in the concentration range of 6.25–62.5 µg/mL is necessitated because of the significant loss of propofol in the perfusion system and during passage through the lung.

To determine whether the oxidative injury that resulted in ACE-shedding or the effects of propofol were directly on the endothelium lining the pulmonary microvessels, a cell culture model system of ACE shedding was developed. We used a stable Chinese Hamster Ovary (CHO) cell line expressing full-length human ACE cDNA that shows a homogeneous pattern of ACE expression and ACE activity similar to that found in endothelial cells in vivo (19). CHO-ACE cells were grown in 100-mm diameter dishes in Ham’s-F12 culture medium supplemented with 2 mM L-glutamine, antibiotic-antimycotic, 20 mM HEPES buffer, 10% fetal bovine serum and 200 µg/mL antibiotic geneticin (G418). For all experiments, the cells were subcultured using trypsin-EDTA onto 96 well plates.

CHO-ACE cells grown on 96-well plates and were washed 3 times with Hank’s balanced salt solution (HBSS) and incubated with propofol or intralipid vehicle diluted in serum-free complete culture medium containing 0.1% BSA (Mediatech, Herndon, VA). After 4 h, the culture fluid was collected. To determine membrane-bound ACE activity and the rate of ACE release (which quantifies ACE activity released into the culture medium relative to membrane-bound ACE activity), cells were lysed with 100 µL of 8 mM 3-[(3-Cholamidopropyl)-dimethylammonio]-1-propanosulfonate (CHAPS). Both culture medium and cell lysates were centrifuged, and ACE activity was determined using a fluorometric assay as described (20).

All compounds used in this study (H2O2, propofol, and intralipid) were tested for cytotoxicity on CHO-ACE cells using a lactate dehydrogenase (LDH) assay kit from Promega (Madison, WI) according to the manufacturer recommendations. Briefly, cells were incubated with different concentrations of H2O2, propofol, and intralipid for 4 h. Then, 50-µL aliquots of the culture medium were transferred to a fresh 96-well plate containing 50 µL reconstituted substrate and incubated for 30 min in the dark. A stop solution (50 µL) was added to terminate the reaction and the absorbance was recorded at 490 nm. Bovine heart LDH was included as a positive control. To determine total LDH levels, cells were lysed by adding 10 volumes of lysis solution from the assay kit and assayed as described above.

Aliquots of culture medium or perfusate were added to 200 µL of 5 mM Hippuryl-His-Leu (Hip-His-Leu) or Z-Phe-His-Leu ACE substrates and incubated for 2 h at 37°C. The reaction was terminated with 0.28 M NaOH and the His-Leu product was estimated fluorometrically as described (20).

CHO-ACE cells were grown in 96-well microtiter plates. Cells were washed three times with HBSS and incubated with various concentrations of H2O2. After washing, cells were fixed with 4% paraformaldehyde for 15 min at room temperature and washed several times with phosphate-buffered saline (PBS). Control mouse immunoglobulin G or 10 µg/mL anti-ACE monoclonal antibodies (mAbs) in PBS + 2% nonfat dry milk were added and incubated for 2 h on ice. Bound mAbs (which reflects membrane-bound ACE) were quantified by incubation with anti-mouse Ab conjugated with alkaline-phosphatase followed by spectrophotometric analysis at OD405.

After perfusion, the lungs were removed from the chamber and blotted with filter paper. The extraneous bronchial and cardiac structures were dissected away and the wet weight of the lung tissue was recorded. To determine the dry weight, the lungs were incubated in a drying oven for 48 h at 60°C and then re-weighed. Lung wet-to-dry weight ratio was calculated using the formula (wet weight – dry weight)/dry weight.

All data are presented as mean ± sd. Statistical comparisons were made using one-way analysis of variance and nonparametric Mann-Whitney U-test. P values < 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
We used the rat IPL to evaluate the effect of different anesthetics on microvascular injury during I/R. Figure 1 demonstrates the kinetics of the ACE appearance in the perfusate after lung isolation and perfusion ex vivo. We observed a steady increase in perfusate ACE activity from 10.7 ± 1.3 mU to 144.1 ± 2.4 mU at 90 min and 167.5 ± 4.8 mU at 2 h in continuously perfused control lung preparations. Pulmonary artery pressure remained stable during the perfusion period. After I/R, ACE release into the perfusate significantly increased after 30 min of ischemia from 123.5 ± 9.3 to 224.9 ± 10.5 mU (82% increase) of ACE in the total perfusate. ACE activity in the perfusate of rat lungs exposed to 30 min ischemia and 30 min reperfusion was 34% more than control lungs. The pulmonary artery pressure was slightly increased in the I/R group immediately after the period of ischemia but returned to preischemia values within the first 5 min after reperfusion (Fig. 1).



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  		Figure 1. Top, the angiotensin-converting enzyme (ACE) release in the isolated perfused lung during ischemia/reperfusion (I/R). ACE activity was measured in the perfusate after control perfusion and after I/R. The data are presented as mU of ACE activity (mean ± sd) in the total perfusate (50 mL); n = 4. Middle, representative recording of pulmonary artery pressure. Lungs and heart were dissected and transferred en bloc to a temperature-controlled chamber. Ventilation was continuous (30% oxygen). Bottom, effect of anesthetics on ACE shedding during I/R. ACE activity in the perfusate was measured spectrofluorometrically using Z-Phe-His-Leu as a substrate. ACE shedding in the perfusate as a result of I/R injury was calculated as follows: (ACE activity in total perfusate) – (ACE activity at the end of perfusion)/ACE activity at the end of perfusion. Propofol (6.25 µg/mL) was compared to a fat emulsion (intralipid) equivalent in concentration to that of the propofol vehicle (*P < 0.05; n = 4) and the inhaled anesthetic isoflurane (2.5%). As a control, we used ACE release from the isolated perfused lung after I/R but without addition of anesthetics or intralipid.

 

After characterizing the I/R model in terms of ACE shedding kinetics, the effect of propofol (6.25 µg/mL) or its vehicle, intralipid, on I/R-induced ACE shedding and lung microvascular injury was assessed and compared with I/R-induced ACE shedding in the presence of the volatile inhaled anesthetic isoflurane (2.5%). Pulmonary artery pressure recorded continuously during each experiment (Fig. 1) indicated there was no significant difference in PP (e.g., from a vasodilatory effect) among the anesthetics used in this study either during the control perfusion period or after I/R. The lung wet-to-dry ratio increased from 4.93 ± 0.35 in control lungs to 6.53 ± 0.90 after I/R (P < 0.05). However, no significant differences were observed in lung wet-to-dry or lung-to-body weight ratios among groups presented in Fig. 1—I/R, I/R with inhaled anesthetic, or I/R with propofol or intralipid (data not shown).

To specifically address endothelial injury, we measured the rate of ACE shedding into the perfusate in the rat IPL during I/R. Data were calculated as reperfusion/perfusion ratio (R/P): (ACE activity in total perfusate) – (ACE activity at the end of perfusion)/ACE activity at the end of perfusion (Fig. 1). For the propofol group, the R/P ratio was equal to 76.6% ± 6.5% of the value determined in the intralipid control group (P < 0.05). Isoflurane did not change ACE release after reperfusion compared to control.

To study the effect of propofol on oxidative lung injury, we measured the effect of H2O2 on pulmonary endothelial ACE release in the rat IPL. In control lungs, ACE activity released into the perfusate steadily increased during the period of perfusion. Figure 2 shows that H2O2 caused a dose-dependent increase in perfusate ACE activity. We observed a significant increase in ACE shedding as rapidly as 30 min after adding H2O2 (168% ± 14% and 304% ± 64% of control with 0.5 mM and 0.75 mM H2O2, respectively) (P < 0.05 versus control; n = 6). At the largest concentration of H2O2 tested (1 mM), there was a dramatic release of ACE into the perfusate and massive edema formation within 50 min of addition of H2O2. The marked increase of ACE release induced by H2O2 was accompanied by an increase in pulmonary artery pressure and an increase in lung wet-to-dry weight ratio of 2.7 times (274% ± 19% of control, P < 0.001, n = 4). Based on these data, we used 0.75 mM H2O2, which caused marked increase in ACE activity but did not result in pulmonary edema (wet-to-dry weight ratio equal to 115% ± 9.2% of control) to evaluate the effect of propofol on H2O2-induced oxidative injury.



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  		Figure 2. Top, concentration-dependent angiotensin-converting enzyme (ACE) release in isolated perfused lungs (IPLs) induced by H2O2. After an equilibration period, lungs were perfused with Krebs-bovine serum albumin (BSA) containing indicated concentrations of H2O2 for 2 h. Data presented as mean ± sd, n = 4. *P < 0.05 compared with control. Bottom, effect of propofol on the ACE release into perfusate induced by H2O2 in IPL. Lungs were perfused with Krebs-BSA (control) versus Krebs-BSA containing 0.75 mM H2O2. In another set of animals, intralipid or propofol were added to the Krebs-BSA buffer with or without H2O2. *P < 0.05 in comparison with fat emulsion. Data presented are mean ± sd, n = 4.

 

To evaluate the effect of propofol, lungs were perfused with either 1x or 10x intralipid or propofol in the presence or absence of 0.75 mM H2O2 for 2 h. Neither intralipid nor propofol per se at 1x (Fig. 2) or 10x (not shown) concentrations changed the rate of ACE release into the perfusate. Perfusion of rat lungs with 1x (Fig. 2) or 10x intralipid (not shown) in the presence of H2O2 caused a similar increase in ACE activity as perfusion with H2O2 alone. The lung wet-to-dry weight ratio did not change. In contrast, perfusion of propofol at 1x concentration caused a significant attenuation of H2O2-induced ACE release into the perfusate (approximately 50%, P < 0.05) compared with intralipid (Fig. 2). A larger concentration of propofol (10x, 62.5 µg/mL) and smaller concentration (1 µg/mL) reduced ACE shedding to the same extent as 1x propofol (data not shown).

To confirm the effect of propofol on ACE release demonstrated in the IPL model, we used a CHO-ACE cell line (clone 2C2) that expresses full-length human ACE at levels similar to that observed in the lung vasculature (19). To induce oxidative injury, the cells were treated with different concentrations of H2O2. Figure 3 shows the gradual increase in ACE release from the cell surface into the medium with increasing H2O2 concentration. There was no significant effect on ACE release at H2O2 concentrations of 0.5 and 1 mM, but 2.5 mM increased ACE release into the medium by 174% ± 10% over untreated control cells.



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  		Figure 3. Top, effect of H2O2 on angiotensin-converting enzyme (ACE) release from the cell surface. Chinese Hamster Ovary (CHO)-ACE cells were grown in 96-well plates in Ham’s F12 containing 10% fetal bovine serum (FBS). The cells were washed 3 times with Hank’s balanced salt solution (HBSS) to remove residual ACE and incubated for 4 h in 200 µL of serum-free "complete" medium with and without different concentrations of H2O2. After that, culture medium was collected and centrifuged, and ACE activity in 50-µL aliquots was measured using Z-Phe-His-Leu as a substrate. Bottom, effect of propofol on H2O2-induced ACE release from the cell surface. CHO-ACE cells were incubated for 4 h in 200 µL of serum-free "complete" medium containing intralipid or propofol at 1x and 10x concentration with and without 2.5 mM H2O2. Culture medium was then analyzed for an enzymatic activity of ACE released from the cell surface. *P < 0.05 in comparison with control (n = 4).

 

To fully characterize this model and to address the issue of the mechanism of increased ACE release in CHO-ACE cells after H2O2 treatment, we measured the level of ACE expression on the surface of CHO-ACE cells treated with H2O2 by two different assays: i) Cell ELISA, using a panel of mAbs against ACE (19), and ii) ACE activity by enzymatic assay. Figure 4 shows no significant increase in surface ACE expression as reflected by anti-ACE mAb binding to CHO-ACE cells treated with 0.5 mM H2O2, whereas treatment of cells with 1.0 or 2.5 mM H2O2 significantly increased the binding of all anti-ACE mAbs tested. The mean increase in mAb binding (for all 8 mAbs tested) with 2.5 mM H2O2 was 232% ± 94% (P < 0.05 compared with untreated CHO-ACE cells). Cytotoxicity tests revealed that 2.5 mM but not 0.5 or 1 mM H2O2 induced LDH release from the cells, which exceeded 10 times the spontaneous LDH release. Nevertheless, the increase in mAb binding was not the result of membrane permeabilization. As proof, CHO-ACE cells permeabilized with 0.1% Triton X-100 (21) showed a similar pattern of mAb binding at all H2O2 concentrations compared to non-permeabilized cells (data not shown). The membrane bound ACE activity was also increased after treatment of CHO-ACE cells with H2O2 (120% ± 6%, P < 0.05), although the magnitude of the increase was less than that determined by cell ELISA using anti-ACE mAbs.



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  		Figure 4. Effect of H2O2 on angiotensin-converting enzyme (ACE) expression on the surface of Chinese Hamster Ovary (CHO)-ACE cells. CHO-ACE cells were grown in 96 well plates. The cells were washed 3 times with Hank’s balanced salt solution (HBSS) to remove residual ACE and incubated for 4 h in 200 µL of serum-free "Complete" medium with or without different concentrations of H2O2. After washing, the cells were fixed with 4% paraformaldehyde, washed and incubated with a panel of monoclonal antibodies (mAbs) to ACE and control mouse non-immune immunoglobulin G (10 µg/mL) diluted in phosphate-buffered saline/2% nonfat dry milk. Bound antibodies were revealed with anti-mouse polyclonal antibodies conjugated with alkaline phosphatase. Background absorbance, in the presence of non-immune mouse immunoglobulin G, was subtracted from each value. *P < 0.05 compared with control.

 

Thus, we conclude that treatment of CHO-ACE cells with H2O2 leads to increase surface ACE expression and a measurable increase in ACE shedding. This system allowed us to study the effect of different compounds on the process of proteolytic release of ACE from the surface of these cells, which represents (at least in terms of surface ACE expression) a convenient in vitro model of lung endothelial cell injury.

Next, we investigated the effect of propofol and intralipid on ACE release from the surface of these ACE-expressing cells. Figure 3 shows that intralipid at 1x and 10x concentrations, and propofol at 1x concentration, did not affect ACE shedding, whereas 10x propofol increased ACE shedding by approximately 50%. Propofol showed a slight cytotoxic effect (LDH release) only at the 10x concentration, which was at least 6 times less than that induced by 2.5 mM H2O2. Figure 3 demonstrates the protective effect of propofol on H2O2-induced ACE release from the cell surface. Despite the fact that 10x propofol induced ACE release by itself (Fig. 3), it diminished ACE shedding induced by 2.5 mM H2O2 from 221% ± 23% to 162% ± 7% of control (P < 0.05; n = 4). A protective effect of 1x or 10x intralipid or 1x propofol was not observed on H2O2-induced ACE release.


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
Propofol, a widely used IV anesthetic, is structurally similar to vitamin E and has been shown to have similar antioxidant properties (22). We focused on the potential of propofol to protect against I/R (oxidative) lung injury that typically accompanies lung transplantation, CPB, and after lung resection. Lung ischemia stimulates the generation of oxidants by pulmonary endothelial cells, which leads to auto-oxidative damage (1). Thus, the aim of this study was to test the hypothesis that propofol protects against I/R-induced acute lung injury in the isolated perfused rat lung. Oxidative injury was assessed by measuring lung wet-to-dry weight ratio and ACE shedding from pulmonary microvascular endothelial cells. We also investigated the effect of propofol on H2O2-induced ACE shedding in a cell culture model system in which ACE expression and shedding can be routinely quantified and experimental treatment protocols tightly controlled.

ACE is an early marker of underlying pulmonary capillary endothelial dysfunction (12,13,23,24). Both I/R and hydrogen peroxide induced a marked increase in ACE release from the endothelial surface of the rat IPL. ACE (CD143) is a membrane glycoprotein that is constitutively expressed on the surface of lung microvascular endothelium (25). Most of the ACE molecule is exposed to the bloodstream at the luminal surface of endothelial cells and thus is highly susceptible to damaging agents, including oxygen radicals. Importantly, ACE content in the pulmonary vasculature is markedly more than that in the systemic vasculature (25). These properties make ACE a very sensitive indicator of lung microvascular endothelial dysfunction or membrane damage (12,13,23,24).

In the present study, putative protective effects of propofol were tested using ex vivo and in vitro model systems. The protective effect of propofol on ACE shedding (a sensitive marker of oxidative injury of endothelial cells) demonstrated in our ex vivo IPL preparation was confirmed in the in vitro cell culture system. All studies were performed at an early stage of lung injury when increased ACE release indicating endothelial dysfunction of pulmonary microvasculature was observed, although changes in lung wet weight and the pulmonary edema formation had not yet occurred.

Our data on ACE release in the isolated lung resulting from I/R or H2O2 treatment are in accordance with previously published studies (12,13). In the rat lung model, we observed diminished release of ACE from the lung vasculature induced by I/R in the presence of propofol as compared with intralipid (Fig. 1). The mechanism of lung dysfunction as a result of reperfusion after ischemia is multifactorial. One of the components of I/R-induced injury is the release of oxygen-derived free radicals (1). The protective effect we observed on I/R microvascular injury probably relates to the antioxidant capacity of propofol rather than to its anesthetic properties, as the tested volatile anesthetic isoflurane did not affect ACE release after I/R. In contrast to these findings, Liu et al. (10,11) demonstrated a protective effect of isoflurane and sevoflurane on I/R-induced injury in isolated rabbit and rat lungs. The differences in the study design and species used may have contributed to the observed differences in the protective effect of volatile anesthetics in the above mentioned studies. Thus, Liu et al. (11) investigated the rabbit IPL, and therefore species differences may have contributed to this discrepancy. However, these authors also demonstrated that isoflurane and sevoflurane attenuated I/R-induced lung injury in the rat IPL (11). The differences in experimental design between our study and Liu et al. (11) are as follows: i) equilibration period: 5 min in our study, 30 min in (11); ii) ischemia: 30 min in our study, 60 min in (11); ii) reperfusion: 30 min in our study, 60 min in (11); iii) perfusion buffer: 3% BSA in our study, 5% BSA, 0.1% dextrose, and 0.2 mU insulin in (11). Moreover, Liu et al. (11) introduced three different anesthetics into the preisolation procedure (barbiturate, ketamine, and lidocaine). In contrast, our strategy was to try to eliminate any "cross-talk" conditions that multiple anesthetics may produce. Admittedly, in the case of propofol-treated lungs, isoflurane was used for induction and surgery (5–7 min). However, we believe this short duration isoflurane exposure did not have a measurable influence on our results.

With H2O2-induced endothelial injury, ACE release increased compared with control (not treated with H2O2). The rate of ACE release, in comparison with control perfusion, was very fast during the first 30 min and then the slope of the curves declined (Fig. 2), which probably relates to the degradation of H2O2 in the perfusate. Propofol attenuated ACE release from the lung vasculature as compared with intralipid control (Fig. 2). In support of our data, it was shown recently that propofol also relieves endotoxin-induced lung injury in rabbits (26).

In the in vitro system, CHO cells transfected with human ACE cDNA were used as a model of lung endothelial cell ACE release. As we described previously, CHO-ACE cells demonstrate a spontaneous proteolytic release of ACE from the plasma membrane (ACE shedding) with a rate similar to that shown for endothelial cells (19,27). We found that treatment of CHO-ACE cells with H2O2 caused a dose-dependent burst in ACE shedding from the surface of CHO-ACE cells (Fig. 3). At the same time, the cells treated with H2O2 revealed a dose-dependent increase in binding of anti-ACE mAbs (Fig. 4; cell ELISA). The mean increase in anti-ACE mAb binding was 2.3-fold at 2.5 mM H2O2. Although ACE activity in the cells treated with this concentration of H2O2 increased only to 120% ± 6% (P < 0.05), the cell ELISA data may more correctly reflect the real increase in ACE expression, as H2O2 tends to inactivate, at least to some extent, ACE (28,29). H2O2 can function as a signaling molecule to activate gene expression. For example, H2O2 induced up-regulation of ICAM-1and P-selectin expression in endothelial cells (30,31).

We speculate that an increase in ACE activity in the culture medium reflects the increase in ACE shedding from the cell surface as a result of an increase in ACE expression and an activation of a proteolytic cascade induced by apoptosis signals. In our studies, 2.5 mM H2O2 caused cell membrane permeabilization and release of LDH into the culture medium. Influx of calcium into these permeabilized cells from the culture medium could also account for the increased rate of ACE release from the cell surface. Our previously published data agree with reports showing the activation of solubilizing proteases in response to calcium influx (27,32).

We found that large-dose propofol diminished H2O2-induced ACE release from the surface of CHO-ACE cells and that this effect occurred despite the fact that propofol per se at that concentration caused an increase in ACE shedding resulting from a cytotoxic effect as determined by a parallel increase in LDH release. The protective effect of propofol observed in cell culture is probably related to the ability of this drug to act as a scavenger of free radicals and inhibitor of Ca2+ channels. The antioxidant and radical scavenging properties of propofol were shown previously in many systems (2–5). The protective effect of propofol on erythrocytes during CPB was also attributed in part to its ability to prevent intracellular Ca2+ overload (33).

In conclusion, we evaluated the effect of propofol on I/R and H2O2-induced lung endothelial injury using the rat IPL and ACE-expressing CHO stable cell line. Perfusion of the rat lung with propofol attenuated the release of ACE from the pulmonary endothelial bed induced in both models. Propofol also decreased the rate of ACE release from the cell surface of CHO-ACE cells. We speculate that propofol may diminish lung endothelial dysfunction in patients suffering from I/R-related oxidative insults (lung transplantation and CPB surgery).

The authors thank Professor Ronald F. Albrecht, Head of the Department of Anesthesiology, University of Illinois at Chicago for his encouragement and support of this project.


   Footnotes
 
Presented, in part, at the 2003 annual meeting of the American Society of Anesthesiology, October 11–15, 2003, San Francisco, California.

Accepted for publication September 28, 2004.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Fisher AB, Dodia C, Tan Z, et al. Oxygen-dependent lipid peroxidation during lung ischemia. J. Clin. Invest 1991;88:674–9.
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