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Anesth Analg 2007; 105:1363-1370
© 2007 International Anesthesia Research Society
doi: 10.1213/01.ane.0000281144.06703.0d
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CRITICAL CARE AND TRAUMA

Propofol Attenuates Endotoxin-Induced Endothelial Cell Injury, Angiotensin-Converting Enzyme Shedding, and Lung Edema

E. Gina Votta-Velis, MD*, Richard D. Minshall, PhD*{dagger}, David J. Visintine, BA*{dagger}, Maricela Castellon, BS*{dagger}, and Irina V. Balyasnikova, PhD*

From the Departments of *Anesthesiology and {dagger}Pharmacology, University of Illinois College of Medicine, Chicago, Illinois.

Address correspondence and reprint requests to Irina V. Balyasnikova, PhD, Anesthesiology Research Center, University of Illinois at Chicago, 1819 W. Polk St. (M/C 519), Chicago, IL 60612. Address e-mail to irinabal{at}uic.edu., irinabal{at}gmail.com.


   Abstract
Top
 Abstract
Introduction
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
BACKGROUND: Acute lung injury (ALI) is a frequent complication in septic patients. Previously, we found that propofol, a highly lipid-soluble anesthetic, attenuates ischemia-reperfusion and oxidative lung injury in the isolated perfused rat lung. In the present study, we evaluated the effect of propofol on endotoxin-induced ALI and endothelial dysfunction.

METHODS: The effect of propofol on endotoxin-induced lung endothelial injury was evaluated by plasma and lung tissue homogenate angiotensin I converting enzyme (ACE) activity, pulmonary vascular anti-ACE monoclonal antibody binding, and lung wet weight to body weight ratio (LW/BW).

RESULTS: When injected IV into rats, endotoxin produced endothelial cell injury and lung edema, as indicated by: 1) an increase in plasma ACE activity, 2) a decrease in lung ACE activity and anti-ACE monoclonal antibody binding, and 3) an increase in LW/BW. Monoclonal antibody 1A2 was up to 1.8 times more sensitive than other anti-ACE monoclonal antibodies in detecting the decrease in ACE in lungs of endotoxin-treated rats. Pretreatment of rats with a bolus of propofol before endotoxin injection significantly inhibited the increase in ACE activity in the blood, the decrease in ACE activity in the lung, the decrease in anti-ACE monoclonal antibody binding in the lung, and the increase in LW/BW ratio. Importantly, propofol also significantly increased the survival rate of endotoxin-treated animals. The protective effect of propofol in endotoxin-treated animals in vivo was confirmed in vitro, i.e., propofol decreased endothelial cell injury and ACE shedding from endothelial cells in culture.

CONCLUSIONS: These results suggest that propofol offers significant protection against endotoxin-induced pulmonary microvessel endothelial cell injury and that anti-ACE monoclonal antibody 1A2 is a sensitive probe for monitoring endothelial dysfunction and ALI during sepsis.


   Introduction
Top
 Abstract
 Introduction
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Angiotensin I-converting enzyme (ACE) is an important component of arterial blood pressure regulation (1). ACE is expressed on the luminal surface of endothelial cells of different types of blood vessels (2). The pulmonary circulation is enriched with capillaries, 100% of which express ACE (3,4). Changes in ACE activity in lung tissue, serum, and bronchoalveolar lavage fluid have been reported during pathological conditions in the lung (5). Both clinical and experimental results show that injury to the lung endothelium is accompanied by a decrease in ACE activity in the lung homogenate, with a transitory increase in its activity in the serum (6–9). Therefore, ACE is an early and sensitive marker of lung endothelial dysfunction (6,10,11).

It has been shown that after systemic injection, anti-ACE monoclonal antibody 9B9 selectively accumulates in the lungs of several mammals (12,13), including humans (14). It also has been reported that monoclonal antibody 9B9 lung uptake is significantly decreased in endotoxin-challenged rats (15), and that radiolabeled monoclonal antibody 9B9 is a more sensitive probe when compared with other markers of pulmonary damage (ACE activity, products of lipid peroxidation) and lung edema (accumulation of serum proteins and edema fluid) (15).

Acute lung injury (ALI) is a frequent complication in septic patients. Reactive oxygen species (ROS), nitric oxide (NO), and proinflammatory cytokines are only a few of the many mediators of sepsis-associated ALI. Previously, we found that propofol, a lipid-soluble anesthetic, attenuates ischemia-reperfusion injury and oxidative-lung injury in the isolated perfused rat lung (16).

Propofol has several properties which indicate that it might be beneficial in ALI. For example, it is structurally similar to vitamin E and has similar antioxidant properties (17), and it acts as a scavenger of peroxynitrite and oxygen-free radicals (18,19). Propofol also inhibits platelet aggregation in vitro and in vivo, inhibits neutrophil function (20,21), and reduces the production of antiinflammatory cytokines in endotoxin-treated animals (22).

We developed a new panel of monoclonal antibodies to rat ACE that selectively accumulate in the lung after systemic injection (23). Several of these antibodies have improved properties, such as increased lung uptake and one order of magnitude higher lung specificity, when compared with the well characterized anti-ACE monoclonal antibody 9B9 (23). In the present work, we compared the biodistribution of these anti-rat ACE monoclonal antibodies in endotoxin-challenged rats to find a sensitive probe for evaluation of lung endothelial dysfunction. Using monoclonal antibody 1A2 binding in vivo in comparison with other criteria of lung injury and endothelial dysfunction, we determined the effect of propofol in endotoxin-treated rats and cultured endothelial cells.


   METHODS
Top
 Abstract
 Introduction
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animal Protocol
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. Under anesthesia, the rats were tracheally intubated and maintained on 2% isoflurane in oxygen. The animals were then injected IV with either 0.5 mL saline or a bolus of IV lipid emulsion or propofol (10 mg/kg) (22). After 1 h, all animals were injected IV with either saline or endotoxin (lipopolysaccharide, LPS; Sigma Chemical Company) at 2 mg/kg and allowed to recover. Radiolabeled antibodies (1 µCi in 0.5 mL of saline) were injected IV via tail vein 16 h after LPS or saline injection. After 1 h, the animals were killed, blood was collected, and internal organs were excised, washed in saline, blotted, and weighed. Tissue radioactivity was determined by gamma scintillation counting. Results are expressed as percent of injected dose per gram of tissue, which reflects the efficiency of antibody accumulation.

Antibody Radiolabeling
Radioiodination of antibodies with 125I was performed in Iodo-Gen precoated tubes (Pierce, Rockford, USA) per manufacturer's instructions as previously described (23). Excess iodine was removed by gel-filtration on a PD-10 (Sephadex G-25) mini-column (Pharmacia, Uppsala, Sweden).

ACE Activity Assay
ACE activity of plasma and lung homogenates was measured using ACE substrate (2 mM Z-Phe-His-Leu or 5 mM Hip-His-Leu) as described previously (24). The fluorescence of samples was measured using 365 nm excitation/500 nm emission filters.

Lung Wet Weight-to-Body Weight Ratio
The body weight of animals was determined before the experiment. Sixteen hours after LPS injection, animals were killed and the lungs were removed and blotted with filter paper. The extraneous bronchial and cardiac structures were dissected away and the wet weight of the lung tissue was recorded. The ratio of lung wet weight-to-body weight ratio (LW/BW) was used as an index of pulmonary edema formation (15).

Biodistribution Monoclonal Antibodies 9B9, 1A2, 2E1, and 4H3 in LPS-Treated Animals
We previously reported that different monoclonal antibodies to ACE selectively, but with differing specificities, accumulate in rat lung tissue after systemic injection (23). It has also been shown that endotoxin causes a decrease in the lung accumulation of monoclonal antibody 9B9 (15). To determine the variability in biodistribution of monoclonal antibodies to rat ACE, we simultaneously compared monoclonal antibodies 9B9 (kindly provided by Dr. Danilov, University of Illinois at Chicago), 1A2, 4H3, and 2E1 in control and endotoxin-treated rats. This allowed us to choose the best and most sensitive system for monitoring the degree of lung injury in rats, using the changes in monoclonal antibody accumulation as a sensitive measure.

Effect of Propofol on Monoclonal Antibody 1A2 Lung Uptake in LPS-Treated Animals
In these experiments, we used monoclonal antibody 1A2 for detection of LPS-induced lung ACE shedding. All animals were divided into four groups: control, propofol alone, LPS alone, and propofol/LPS.

Effect of Propofol on Plasma and Tissue ACE Activity, Edema Index, and Survival Rate in LPS-Treated Animals
To validate the results on tissue ACE expression obtained by measuring 125I-labeled monoclonal antibody 1A2 lung accumulation, we directly measured ACE activity in lung homogenates and plasma of these animals. In addition to ACE measurements, we calculated the LW/BW ratio as an index of pulmonary edema formation.

Cell Culture
ACE expression on the plasma membrane of primary culture endothelial cells decreases with propagation and passage (25). Therefore, to study the effect of LPS on endothelial cell ACE expression in vitro, we generated a stable rat lung microvascular endothelial-hACE cell line, which had no detectable ACE on the surface before transfection (26), by transducing full-length wild-type human ACE cDNA (27). The initial population of hACE-transfected rat lung microvascular endothelial cells was selected using 600 µg/mL geneticin (G418). The selected population was subcloned by dilution cloning at a density of one cell per well in 96-well plates, and cells were grown until individual colonies were distinguishable. Approximately 10 days later, wells containing individual clones were washed, and media was replaced with serum-free media. Media collected from individual clones over 24 h was analyzed for ACE activity. Based on this criterion, several clones were selected and further expanded. Of the stable hACE-expressing rat lung microvascular endothelial cell lines generated, clone 1C10, which showed a homogeneous pattern of ACE expression and activity similar to that found in endothelial cells in vivo (25), was used for all in vitro experiments. Rat lung microvascular endothelial-hACE cells were grown in 100-mm diameter dishes in DMEM culture medium supplemented with 2 mM l-glutamine, antibiotic-antimycotic, 10% fetal bovine serum, and 200 µg/mL G418. For all experiments, the cells were subcultured using trypsin-EDTA onto 96-well plates.

ACE Shedding and Lactate Dehydrogenase Assay
Rat lung microvascular endothelial-hACE cells grown in 96-well plates were washed three times with Hank's balanced salt solution and incubated with LPS (0.1 or 10 µg/mL) or LPS together with propofol (1 or 5 µg/mL) diluted in serum-free medium. After 24 h, the culture fluid was collected and centrifuged at 1500 rpm for 10 min, and ACE activity was determined using a fluorometric assay, as described above.

Lactate dehydrogenase (LDH) release from rat lung microvascular endothelial cells was used as an additional marker of cell injury using an assay kit from Promega (Madison, WI). LDH in the culture medium collected from rat lung microvascular endothelial-hACE cells after 24 h incubation with LPS or LPS/propofol was estimated as recommended by the manufacturer. Bovine heart LDH was included as a positive control.

Cell Enzyme-Linked Immunosorbent Assay
The level of expression of human ACE on the surface of rat lung microvascular endothelial-hACE cells treated with various concentrations of LPS or LPS/propofol for 24 h was estimated in cell enzyme-linked immunosorbent assay using the anti-ACE monoclonal antibody i2H5 (kindly provided by Dr. Danilov, University of Illinois at Chicago), as previously described (23).

Statistical Analysis
All data are presented as mean ± sd. Statistical comparisons were made using Student's t-test or one-way analysis of variance, followed by the Bonferroni post hoc test. P values <0.05 were considered significant.


   RESULTS
Top
 Abstract
 Introduction
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Comparison of Biodistribution Monoclonal Antibodies 9B9, 1A2, 2E1, and 4H3 in LPS-Treated Animals
Figure 1A shows that the accumulation of monoclonal antibodies 9B9, 1A2, 4H3, and 2E1 in control animals was 28 ± 1.59, 47 ± 0.46, 40 ± 3.04, and 47 ± 0.4%ID/g (mean ± sd; n = 7), respectively. The percent accumulation of all monoclonal antibodies was significantly decreased by an average of 1.5 times in LPS-treated animals (P < 0.05). We found that monoclonal antibody 1A2 showed the largest difference between control and LPS-treated animals with respect to lung accumulation, at 54% ± 4.7% of control (n = 7), whereas monoclonal antibodies 9B9, 4H3, and 2E1 showed 69.2% ± 6%, 75% ± 5.4%, and 68.4% ± 8.1% accumulation in LPS-treated rats compared with untreated controls (100%), respectively (n = 7) (P < 0.05) (Fig. 1B). Thus, LPS caused a significant decrease in anti-ACE monoclonal antibody accumulation, i.e., in ACE content in the lung endothelium. Antibody biodistribution in other organs was not affected (data not shown).


Figure 131
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  		Figure 1. Anti-angiotensin-converting enzyme (ACE) monoclonal antibody binding in rats treated with lipopolysaccharide (LPS). Radiolabeled monoclonal antibodies (1 µCi) were injected IV 16 h after LPS (2 mg/kg) injection. A, Comparison of anti-ACE monoclonal antibody lung uptake in control and LPS-treated animals presented as percent of injected dose per gram of tissue (%ID/g) of lung tissue. B, Lung uptake of monoclonal antibodies in LPS-treated animals presented as percent of control animals. The data are mean ± sd, n = 7; *-Statistically significant difference in lung antibody uptake between control and LPS groups (P < 0.05).

 

Effect of Propofol on Monoclonal Antibody 1A2 Lung Uptake in LPS-Treated Animals
Figure 2 shows that propofol at a bolus dose of 10 mg/kg did not affect 125I-monoclonal antibody 1A2 accumulation in the rat lung (98% ± 5% of that in the control group, n = 7). In LPS-treated animals, 125I-1A2 lung uptake was significantly decreased (56.5% ± 11.2% of 1A2 lung accumulation in the control group; n = 7, P < 0.05). However, propofol injected 1 h before LPS administration attenuated the decrease in monoclonal antibody 1A2 lung uptake induced by LPS by 44%. The lung uptake of monoclonal antibody 1A2 in this group was 75.5% ± 13% (n = 4) of that in control. Thus, propofol injected 1 h in advance of LPS diminished ACE shedding as measured by monoclonal antibody 1A2 lung uptake.


Figure 231
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  		Figure 2. Effect of propofol on monoclonal antibody 1A2 lung uptake in lipopolysaccharide (LPS)-treated rats. A bolus of propofol (10 mg/kg) was injected 1 h before LPS challenge. After 16 h, radiolabeled monoclonal antibody 1A2 was injected IV in all groups of animals. Lung uptake of monoclonal antibody 1A2 (percent of injected dose per gram of tissue (%ID/g) of lung tissue) is shown. The data are mean ± sd, n = 7; *P < 0.05 (versus control); & P < 0.05 (versus LPS), n = 4.

 

Effect of Propofol on Plasma and Tissue ACE Activity, Edema Index, and Survival Rate in LPS-Treated Animals
ACE activity in plasma and lung homogenates was not affected by a bolus injection of IV lipid emulsion (data not shown). Similar to what was observed when measuring monoclonal antibody 1A2 lung accumulation, propofol per se also did not significantly affect lung ACE activity (and there was no significant increase in plasma ACE enzymatic activity detected in the propofol group compared with control animals) (Figs. 3A and B). In LPS-treated rats, ACE activity in the lungs was significantly decreased and was equal to 36% ± 4.6% (P < 0.05, n = 8) of the activity in control animals (Fig. 3A). The amount of ACE was increased 1.5-fold in the plasma of these animals compared with the amount of ACE in plasma of control animals (Fig. 3B). Pretreatment with propofol reduced the effect of LPS on ACE release from the lung by 37% (49.3% ± 4.1% of propofol control, n = 8). The amount of ACE released into the blood of these animals was diminished approximately two times when compared with the LPS group (Figs. 3A and B). IV lipid emulsion by itself did not affect ACE activity in the plasma (104% ± 12.4%, n = 4) or lung tissue (87% ± 19.8%) in comparison with LPS alone. Thus, propofol partially protected the rat lung with respect to ACE release, an early marker of pulmonary endothelial dysfunction.


Figure 331
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  		Figure 3. Effect of propofol on lung and plasma angiotensin-converting enzyme (ACE) activity and edema index in lipopolysaccharide (LPS)-treated rats. A bolus of propofol (10 mg/kg) was injected 1 h before LPS challenge. After 16 h, radiolabeled monoclonal antibody 1A2 was injected, and the tissues were harvested after 1 h. ACE activity in lung homogenates and plasma was estimated enzymatically using Hip-Hs-Leu as substrate. Edema index was calculated as the lung wet weight to body weight ratio. The data are expressed as percent of control, mean ± sd, n = 8; *P < 0.05 (versus control); and P < 0.05 (versus LPS).

 

The LW/BW ratio in the IV lipid emulsion group (100% ± 4.58%, n = 4) did not differ from that in the control (saline) group of rats (data not shown). Figure 3C shows that the LW/BW ratio in propofol-treated rats was unchanged, in agreement with the data obtained on ACE content in the lung. The ratio significantly increased 1.31-fold in LPS-treated animals compared with the control group. There was no effect of IV lipid emulsion on LW/BW ratio in LPS-treated animals (102% ± 2.86% of LPS alone, n = 3, data not shown). However, this ratio was markedly (54%) reduced in LPS-treated animals that were pretreated with a bolus injection of propofol. Finally, we observed that several animals in the LPS-treated group did not survive, whereas none of the animals pretreated with propofol died after LPS injection. Thus, the survival rate of rats pretreated with a bolus of propofol before LPS injection was significantly better than that of rats treated with LPS alone (100% vs 77%, respectively).

Effect of LPS on Endothelial ACE Content
Clone 1C10 showed a homogeneous pattern of ACE expression as determined by immunocytochemistry (data not shown) and therefore was chosen for all in vitro experiments. The specific activity of ACE ranged from 13 to 26 mU/mg of cell protein, which is similar to that in primary culture of endothelial cells (25).

Figure 4A shows that 24-h treatment of rat lung microvascular endothelial-hACE cells with LPS caused a dose-dependent release of ACE from the cell surface, a decrease in ACE expression on the cell surface, and LDH release from the cells. Maximal release of ACE from the cell surface and the decrease in ACE expression in these cells was observed with 1.66 µg/mL of LPS, and was equal to 180% and 77% of that in untreated cells, respectively. LDH release reached 144% of control at the maximum LPS concentration used.


Figure 431
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  		Figure 4. A, Effect of lipopolysaccharide (LPS) on angiotensin-converting enzyme (ACE) and lactate dehydrogenase (LDH) release and ACE expression. Rat lung microvascular endothelial cells-hACE expressing cells were washed three times with Hank's balanced salt solution to remove residual ACE and incubated for 24 h in 200 µL of serum-free medium with different concentrations of LPS. ACE activity in the supernatant was measured using Z-Phe-His-Leu as substrate. LDH release was estimated using a Promega kit, and the level of ACE expression on the cell surface was estimated by cell enzyme-linked immunosorbent assay. Data are mean ± sd, expressed as percent of control, where control is the cells not treated with LPS. B, Effect of propofol on LPS-induced ACE and LDH release and ACE expression. Rat lung microvascular endothelial-hACE cells were incubated for 24 h in 200 µL of serum-free medium containing propofol at 1 and 5 µg/mL in the presence of various concentrations of LPS. Culture medium was analyzed for ACE enzymatic activity released from the cell surface and for LDH release. The level of ACE expression on the cell surface was estimated using cell enzyme-linked immunosorbent assay and expressed as a percent of control. *Symbol reflects a statistically significant effect of propofol in LPS-treated cells compared with LPS alone (P < 0.05; n = 4).

 

Effect of Propofol on LPS-Treated Endothelial Cells
Propofol at both 1 and 5 µg/mL concentrations did not affect ACE expression on the cell surface (102% ± 4.19% and 100.5% ± 1.74% of control, respectively, n = 4), ACE release (120.1% ± 11.6% and 110.8% ± 29% of control, n = 6), or LDH release (97.8% ± 6.7% and 101.2% ± 8.3% of control, n = 6) into culture media. Figure 4B shows that propofol dose-dependently attenuated ACE release from the cell surface at both concentrations of LPS used. Propofol at a concentration of 1 µg/mL diminished LPS-induced ACE release by 19.4% and was more effective at 5 µg/mL, as ACE release was attenuated by 47.7%. Propofol at a dose of 5 µg/mL preserved ACE expression on the cell surface in cells treated with 1 µg/mL of LPS. However, we found no effect on ACE expression at both 1 and 5 µg/mL of propofol when LPS was used at 10 µg/mL. Finally, propofol was effective, regardless of concentration, at diminishing LDH release from the cells treated with 1 or 10 µg/mL of LPS. Thus, these data demonstrate that propofol prevents LPS-induced ACE and LDH release from endothelial cells, preserving ACE expression and cell viability.


   DISCUSSION
Top
 Abstract
 Introduction
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Septic shock is associated with a high mortality rate in critically ill patients. It is characterized by metabolic acidosis, potentially lethal hypotension, and progressive dysfunction in multiple organs, including the lung (28). Propofol, a lipid-soluble anesthetic, is increasingly used in critically ill septic patients (29). Several publications demonstrate that propofol has a beneficial effect in experimental models of septic shock (30–33).

It has been shown that propofol attenuates the increase in the proinflammatory cytokines tumor necrosis factor {alpha} and interleukin (IL)-6 in endotoxin-exposed rats (32,33). Propofol was also shown to attenuate the increase in lung wet/dry weight ratio, lung injury score, neutrophil accumulation, and concentration of albumin, thromboxane B2, and IL-8 in bronchoalveolar lavage fluid in a rabbit model of endotoxin-induced lung injury (33). Propofol has been shown to increase the survival rate and attenuate the increases in inducible NO synthase (iNOS) and nitrotyrosine expression in lung tissues, pulmonary microvascular permeability, and bronchoalveolar lavage fluid NO2–/NO3– in endotoxin-treated rats (32). However, the mechanism by which propofol attenuates lung endothelial cell dysfunction during ALI is unclear.

In the present study, we demonstrated that injection of endotoxin causes ALI in rats, as revealed by lung edema. To assess lung edema, we used LW/BW ratio, which has been validated in several studies (15,34). We also showed that LPS injection increased ACE activity in rat plasma and decreased ACE activity in the lung. Bolus administration of propofol before endotoxin injection significantly attenuated endotoxin-induced changes, indicating that its administration diminishes injury of lung endothelial cells in endotoxin-treated rats.

The fact that a single bolus of propofol was sufficient to produce a protective effect against endotoxemia is intriguing. Even though propofol is rapidly cleared from the circulation, its effects seem to be long lasting, perhaps due to suppression of mRNA of certain proteins that are increased in the lung after treatment with endotoxin. In support of this hypothesis, it has been shown that the expression of nitrotyrosine in rats pretreated or simultaneously treated with propofol and endotoxin is significantly diminished in comparison with treatment by endotoxin alone (32). Moreover, NO production is reduced because of down-regulation of mRNA for iNOS (32). It has also been shown that propofol has antiinflammatory and antioxidant effects on the biosynthesis of CAT-2B, tumor necrosis factor {alpha}, IL-1ß, IL-6, iNOS, and NO in LPS-activated macrophages, and that this effect is exerted at the pretranslational level (35–37).

In this study, we did not focus on the mechanism by which propofol mediates its protective effect on pulmonary endothelial function in endotoxin-treated rats. It has been reported that lipoproteins have an ability, via surface phospholipids, to bind and neutralize endotoxin, and therefore to improve outcome in animal models of sepsis (38). Nevertheless, the data regarding the effect of IV lipid emulsion, propofol's solvent, on the endotoxin response are very controversial. In vitro, IV lipid emulsion inhibits endotoxin-induced production of cytokines by mononuclear cells (39) and human whole blood (40). On the other hand, it has also been shown that a bolus injection of IV lipid emulsion does not influence the in vivo response to endotoxin in humans (40). The controversy may relate to the different concentrations of IV lipid emulsion used in the various studies, as well as to the variability in human and rodent sensitivity to endotoxin.

We demonstrate here that the improvement in ACE shedding from lung vasculature and the improvement in LW/BW ratio found in the propofol-treated group of animals can be attributed to propofol itself rather than its lipid component. In a previous publication, we demonstrated that lung oxidative injury, which induces ACE release from the surface of lung endothelial cells (15,41), was also attenuated by propofol (16). Therefore, it is possible that the ability of propofol to attenuate ACE release from the pulmonary microvasculature and prevent ALI in endotoxin-treated rats might relate (at least in part) to its antioxidant properties. In support of this concept, the antioxidant property of propofol was demonstrated in an oxidative model of ischemia-reperfusion and hydrogen peroxide-induced lung injury (16).

ACE is a marker of lung endothelial injury (6–11), and anti-ACE monoclonal antibody 9B9 uptake in the pulmonary circulation has been used to monitor lung endothelial ACE expression (15,42). We generated a new panel of anti-ACE monoclonal antibodies to rat ACE with improved selectivity relative to 9B9 (23). In the present study, we compared the biodistribution of anti-rat ACE monoclonal antibodies in endotoxin-challenged rats. Monoclonal antibodies 4H3 and 2E1 showed improved selectivity of lung uptake compared with monoclonal antibody 9B9 (23), but the sensitivity for detecting differences in ACE expression between control and endotoxin-treated animals was similar to monoclonal antibody 9B9. However, monoclonal antibody 1A2 demonstrated a 1.5-fold higher sensitivity for detecting changes in ACE expression, and thus was used in the present studies. The differences in monoclonal antibody-ACE binding may have been due to ACE modification by oxidants and proteases generated locally in LPS-treated animals (43). Herein, we used the lung accumulation of monoclonal antibody 1A2 in rats treated with endotoxin as an alternative method for evaluating lung endothelial dysfunction when compared with measurement of ACE activity in lung tissue and serum.

The mechanism of pulmonary dysfunction due to sepsis is multifactorial. One of the components of LPS-induced injury is the release of ROS from activated neutrophils (44). The protective effect we observed on lung microvascular injury likely relates to the antioxidant properties of propofol. We also reported that propofol diminished the degree of ACE release from the pulmonary circulation in an isolated perfused rat lung model of ischemia-reperfusion and hydrogen peroxide-induced oxidative lung injury (16). The prior studies and the present investigation reproducibly demonstrate that lung accumulation of anti-ACE monoclonal antibodies is significantly reduced in the endotoxin-induced model of septic shock in rats (14,15). This suggests that the measurement of anti-ACE monoclonal antibody binding or accumulation in patients with septic shock may be useful for estimating the degree of lung injury. For example, gamma scintigraphy of radiolabeled anti-ACE monoclonal antibodies (14,45) in septic patients might be used clinically for early detection of potentially fatal pulmonary microvascular injury.

To confirm the effect of propofol on ACE release from the pulmonary circulation demonstrated in the LPS-induced lung injury model in vivo, we used a rat lung microvascular endothelial cell-hACE cell line that expresses full-length human ACE at levels similar to that observed in the lung vasculature. We found that LPS caused the release of ACE from the cell surface of rat lung microvascular endothelial-hACE cells. Our data are in accordance with previous reports on the effect of LPS on ACE activity in cultured human endothelial cells (46,47). In addition, we observed the release of LDH (an indicator of cell death) from rat lung microvascular endothelial-hACE cells in response to LPS treatment, which is consistent with LPS-induced apoptosis (48). Remarkably, propofol diminished the release of ACE and LDH and partially restored ACE expression on cells treated with a low dose of LPS. The mechanism by which propofol attenuates endothelial cell dysfunction, as judged by ACE and LDH release from the cell surface in LPS-treated cells, remains to be elucidated, but may relate to the ability of propofol to scavenge peroxynitrite and ROS (18,19). LPS elicits the generation of ROS (44) and NO (49), which can form peroxynitrite. Consistent with this hypothesis, we previously demonstrated that propofol diminishes ACE release from cells treated with hydrogen peroxide (16).

In conclusion, we found that propofol attenuates endotoxin-induced lung injury in rats in vivo, as shown by 1) the prevention of ACE shedding from the pulmonary circulation, 2) pulmonary edema index, 3) animal survival rate, and 4) endothelial dysfunction in vitro using rat lung microvascular endothelial-hACE cells as a model of the pulmonary endothelium. The results of this study also demonstrate that monoclonal antibody 1A2 raised against rat ACE is a very sensitive tool for detecting ACE in the lung and can be used for monitoring the status of the pulmonary circulation in different models of lung injury in rats. The evaluation of ACE expression on the surface of lung endothelial cells using this approach could be a sensitive method of monitoring endothelial dysfunction and ALI during sepsis.


   ACKNOWLEDGMENTS
 
We thank Dr. Danilov (University of Illinois at Chicago) for providing anti-ACE monoclonal antibodies 9B9 and i2H5 and for critical reading of the manuscript, and Dr. Ronald F. Albrecht, Professor and Head of the Department of Anesthesiology, University of Illinois at Chicago, for supporting this study.


   Footnotes
 
Accepted for publication July 5, 2007.

Presented in part at the annual meeting of the International Anesthesiology Research Society (IARS), Honolulu, HI, 2005.


   REFERENCES
Top
 Abstract
 Introduction
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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