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Am J Physiol Lung Cell Mol Physiol 284: L410-L419, 2003. First published October 25, 2002; doi:10.1152/ajplung.00384.2001
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Vol. 284, Issue 2, L410-L419, February 2003

Mechanical ventilation-induced pneumoprotein CC-16 vascular transfer in rats: effect of KGF pretreatment

Olivier Lesur1, Cédric Hermans2, Jean-François Chalifour1, Johanne Picotte3, Bruno Lévy1, Alfred Bernard2, and Denis Lane1

1 Groupe de Recherche en Physiopathologie Respiratoire et Unité des Soins Intensifs Médicaux, Département de Médecine, 3 Service d'inhalothérapie, Centre Hospitalier Universitaire de Sherbrooke, Quebec, Canada J1H 5N4; and 2 Unité de Toxicologie Industrielle et Médecine du Travail, Université Catholique de Louvain, 1200 Brussels, Belgium


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

After air-blood barrier injury, "pneumoproteins" specific to lung epithelial distal airspaces reaching the bloodstream are putative markers of lung hyperpermeability. The contribution of mechanical ventilation (MV) to this leakage is unknown. To explore this issue, 16-kDa Clara cell protein (CC-16) concentration was quantified in bronchoalveolar lavages (BALFs) and/or sera of rats first exposed either to ambient air or to 48 h of hyperoxia-induced acute lung injury and then ventilated for 2 h according to one of the following strategies: 1) spontaneous ventilation (SV), 2) very-low-volume high PEEP (VLVHP, where PEEP is positive end-expiratory pressure), 3) low-volume zero PEEP, 4) moderate-volume low PEEP, and 5) high-volume zero PEEP (HVZP). Results show that total proteins in BALFs increased with time and MV, with little impact from hyperoxia preexposure. CC-16 content decreased in BALFs but increased in the bloodstream during MV, suggesting intravascular leakage. Lung overdistension may result either from high-volume (HVZP) or high-PEEP (VLVHP) MV, and it was the most potent inducer of CC-16 leakage (P < 0.05 vs. SV). In the VLVHP group, pretreatment with keratinocyte growth factor was efficient in reducing blood CC-16 transfer.

acute lung injury/acute respiratory distress syndrome; ventilation-induced lung injury; 16-kDa Clara cell; keratinocyte growth factor; pneumoproteinemia


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

MECHANICAL VENTILATION (MV) is an important support therapy to patients admitted in intensive care units for respiratory failure. New generations of more sophisticated and computerized respiratory apparatus have allowed considerable progress over the past half-century (7). In addition, there have been tremendous advances in our knowledge of respiratory physiology and pathophysiology under positive-pressure MV. It is now evident that some ventilatory strategies such as high peak and plateau airway pressure, high tidal volume, high respiratory frequency, and high inspiratory flow rate (9, 17, 32, 36, 44), can be deleterious and lead to baro- and/or volotraumas (1). Complementary studies have suggested that inadequate cycling collapse and reopening of distal air spaces are also deleterious but do not exhibit mandatory clinical volo- or barodisaster, although there is macro- and microscopic evidence of induced lung injury. These observations have led to new concepts of ventilation-associated lung injury (in humans) and ventilation-induced lung injury (VILI; in experimental models) along with the term "biotrauma," which has been proposed recently as a novel expression within this framework (13, 25).

Intrapulmonary vascular protein leakage is an essential cogwheel in VILI observations and is also a hallmark of hyperpermeability syndromes, including acute respiratory distress syndrome (ARDS) and acute lung injury (ALI; see Refs. 1, 17, 29). This lung protein leakage allows for the distinction between alterations of the alveolar-capillary (A-C) barrier from high hydrostatic pulmonary edema in which the barrier is intact and in which transfer of proteins into the airspaces is low and oncotically generated (40). All plasmatic proteins can penetrate inside the lungs, but the predominant protein is serum albumin, a medium-sized-molecular-mass molecule. However, recent evidence supports the concept that biological fluid leakage is not exclusively a one-way process but can be also observed moving from the air spaces to the systemic circulation (22). This has definitely been proven by studies measuring proteins specifically produced in lung airspaces and measurable in the circulation, hence the term "pneumoproteinemia" (22). Indeed, lung epithelium-derived small proteins such as surfactant protein (SP)-A, SP-B, 40-kDa rat type I (rTI40)-56-kDa human type I (HTI56) cell-specific proteins, or 16-kDa Clara cell protein (CC-16) have all been tested and validated as blood markers of lung permeability in clinical studies of ALI/ARDS (14, 15, 22, 30, 34). These pneumoproteins could prove to be interesting sensors of lung hyperpermeability either during the natural history of ALI/ARDS or when drug-induced reversal of A-C barrier leakage is to be experimentally tested or clinically administered. In this respect, the fibroblast-derived, epithelial growth-promoting cytokine keratinocyte growth factor (KGF) is a promising molecule shown to efficiently reduce lung injury in several experimental models (45).

Positive-pressure MV can be a source of lung-to-blood transfer of molecules such as recently documented for bacteria in a model of Escherichia coli lung instillation (33), endotoxins (31), and inflammatory mediators (43). MV-induced lung-to-blood transfer of molecules (including pneumoproteins) should a priori be altered by A-C barrier permeability, such as ALI/ARDS, although the contribution of this pneumoprotein transfer as an independent parameter has not been investigated per se.

Hence the main hypotheses of this study are that MV, especially when strategies potentially leading to lung overdistension are set, is a significant modulator of pneumoprotein transfer to the bloodstream in normal and previously injured lungs and that KGF treatment can reduce this leakage. For this purpose, the following parameters were selected: 1) several MV strategies, including high-volume and/or high-pressure setups, 2) hyperoxia-induced cellular damage of the A-C barrier as the archetypal experimental model of lung hyperpermeability (10), 3) CC-16 as a peripheral sensitive marker of lung injury, recently validated both clinically and experimentally (22), and 4) KGF as the agent targeting epithelial repair in the A-C barrier.

Results of this study demonstrate that 1) differentially aggressive ventilation strategies can alter both protein blood and bronchoalveolar lavage fluid (BALF) contents, 2) preliminary alteration of the A-C barrier by hyperoxia can further modulate this bidirectional leakage process with an emphasis on pneumoprotein CC-16 transfer, and 3) reinforcement of the epithelial side of the A-C barrier with KGF can reduce lung CC-16 permeability.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Experimentation was performed in pathogen-free male Sprague-Dawley rats (Charles Rivers Laboratories, St. Constant, PQ, Canada) weighing 325-375 g. All animals received care in compliance with The Guide to the Care and Use of Experimental Animals from the Canadian Council of Animal Care (1993, CCAC, 2nd ed.), and all protocols were approved by our institution's internal animal ethics committee board.

Rats were first anesthetized with xylazine/ketamine (20 and 50 mg/kg im, respectively), with maintenance intramuscular redosing of 50 mg · kg-1 · h-1 when necessary. A tracheostomy was performed with a 14-gauge cannula (Insyte; B-D) inserted and secured to the trachea. The right carotid artery was then denuded, and an angiocatheter (Intramedic polyethylene tubing, ID 0.023 in., OD 0.038 in.; VWR) was introduced for blood sampling and gas analysis and maintained open with heparin saline solution (100 U/ml final concentration). Animals were either left to breathe ambient air or ventilated using a pediatric constant-flow ventilator (infant pressure ventilator BP200; Bourns Life Systems, Riverside, CA). Additional sedation was given with pentobarbital (ip) to either group, when necessary. The average duration of surgical intervention was 20 min, and animal temperature was maintained constant using an electric pad. All surgical and experimental procedures were performed in a supine position, and limb contentions were liberated once surgical cannulations had been performed. Mean arterial pressure (MAP, mmHg) was screened continuously during the experimental period by connecting the arterial catheter to a Guardian Datamedix Monitor through a pressure transducer (PX272; Baxter).

Experimental protocol. Two populations of animals were subjected to multiple ventilatory strategies. The first population consisted of rats left in ambient air with normal unchallenged lungs, whereas the second group was composed of rats exposed to hyperoxia (>95%) for ~48 h in a Plexiglas chamber. A maximum of four rats were simultaneously exposed in a 0.25-m3 confined area equipped with a fan and soda lime; all animals had free access to food and water ad libitum. This model of hyperoxia was previously validated to induce ALI with vascular leak and epithelial apoptosis. Lung injury culminates after 72-96 h of hyperoxia, with massive edema, pleural effusion, and respiratory distress, leading to high mortality rates (39). For this reason, preliminary trials established 48 h as the best time period for this experimental protocol.

Five ventilatory strategies were explored, including one in spontaneous ventilation and four with mechanical support: 1) spontaneous ventilation (SV), 2) very-low-volume high PEEP (VLVHP) MV (where PEEP is positive end-expiratory pressure), 3) low-volume zero PEEP (LVZP) MV, 4) medium-volume low PEEP (MVLP) MV, and 5) high-volume zero PEEP (HVZP) MV. MV strategies were selected to approximate "low-stretch" "open-lung" ventilation (with permissive hypercarbia) for the VLVHP group, "low-stretch" controlled hypoventilation (with hypercarbia) potentially leading to multifocal atelectasis and collapse for the LVZP group, "high-cycling stretch" with end-expiratory collapse for the HVZP group, and a relatively neutral adjustment of ventilatory parameters for the MVLP as a "control" group. These MV setups have been chosen to represent several conditions where combinations of delivered volumes and/or exhibited airway pressures to a given animal can be graded as "nonaggressive" to "aggressive" ventilation according to present knowledge. Furthermore, a very high-volume zero PEEP (VHVZP) group was specifically set up to depict a progress profile of both CC-16 BALF/blood concentrations with increasing tidal volume (Vt) from LVZP and HVZP to VHVZP. All strategies were set up for both populations of normal and hyperoxia-exposed rats. Ventilatory parameters are detailed further in Table 1.

                              
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  		Table 1.   Ventilatory parameters in experimental groups

A steady-state period of 2 h (T2 h) of monitored ventilation was the effective experimental period. T0 is the time point immediately before randomization to SV or MV. Initial plasma and gas samples were taken at T0. Thereafter, a gas analysis was performed every other 30 min, independent of subset assignment, until T2 h for the purpose of parameter adjustment, if needed (Ciba-Corning 238 pH/blood gas analyzer). Normal range values of arterial acid-base balance targeted for all groups (except VLVHP) were as follows: pH, 7.35-7.47; PCO2, 28-42 Torr; HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, 22-30 mmol/l. Arterial acid-base values of VLVHP and LVZP groups differed from the other groups and reached various ranges of values related to mandated ventilatory parameters, i.e., pH, 7.23 ± 0.04; PCO2, 63.8 ± 6.2 Torr; HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, 23 ± 1.8 mmol/l; and pH, 7.2 ± 0.02; PCO2, 60.6 ± 10.9 Torr; HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, 23 ± 0.3 mmol/l, respectively. Oxygen supply by cannula or by the input gas mixture of the ventilator was added to ensure a normal PO2 range of 60-95 Torr. At T2 h, a final gas analysis was performed together with final plasma sampling and a 10-ml PBS bronchoalveolar lavage (BAL; reinfused three times, recovery 7.5 ± 1 ml) before death. T0 BALs were performed separately in subsets of rats both exposed and not exposed to hyperoxia.

Normal saline solution was infused intravenously to replace sampling (300 µl each) and insensible water loss (2 ml/h).

In addition to the core investigation and to assess the impact of epithelial reinforcement in preventing pneumoprotein leakage, a subsequent subset of rats was subjected to intratracheal instillation of 5 mg/kg KGF (provided by Dr. Thomas R. Ulich, Amgen, Thousand Oaks, CA) in 250 µl of vehicle or, in controls, vehicle alone. This instillation was performed immediately before hyperoxia or ambient air exposure and 48 h before the steady-state MV period, selecting the aggressive VLVHP strategy.

Furthermore and in parallel to the above measurements, separate cohorts of rats (not included for protein leakage determination) were used to establish respiratory system pressure volume (PV) curves. PV curves were performed in ambient air and hyperoxia-exposed rats at T0 and T2 h after MV settings, as described by others (8). For this purpose, comparisons were made using the Bourns ventilator and a infant Baby log (8000; Dräger) equipped with a in-line pneumotachograph, both devices exhibiting a constant flow system. Inflation of lungs was performed in 2.5-ml increments to a maximum airway pressure of 30 cmH2O.

Biological fluid processing and analyses. Blood samples collected by the carotid cannula were centrifuged (1,800 g for 8 min at +4°C), and the sera were separated into aliquots and stored at -80°C.

BALFs were processed as described previously (28). Total and differential cell counting was performed and revealed enhanced cellularity with MV (either with or without previous hyperoxia) as follows: five- to eightfold increase of alveolar cells after 2 h of experimentation vs. control rats. Polymorphonuclear neutrophils became predominant in experimental BALFs (from 35 to 50% of total cells in all groups) while representing very few cells in control rats (1.8 ± 1.8%). In addition, after centrifugation, acellular supernatants were frozen and stored at -80°C. Total proteins in BALFs were measured according to the method of Lesur et al. (28). CC-16 concentrations were determined by an automated latex immunoassay, as described previously (21). This assay utilizes rat CC-16 protein purified from concentrated BALFs as a standard, together with a polyclonal antibody raised in rabbit. This antibody was already shown to recognize a single band of ~16 kDa when tested in different biological fluids by Western blot analysis (21). Performances of the assay were similar to that reported for the human protein (21). Analytical recovery averaged 90 ± 7%, and the detection limit was 0.5 µg/l.

Creatinine blood content (µmol/l) was determined in initial and final samplings of experimental rats in an Ektachen multianalyzer using a classic enzymatic method.

Statistical analysis. Data are expressed as means ± SD, with each group comprising six rats. Statistical analyses were performed using ANOVA. Threshold of significance was set at P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Respiratory mechanics allowed establishment of the static compliance of the total respiratory system in our experimental setting. Curves were very similar at T0, either in ambient air- or 48-h hyperoxia-exposed rats (data not shown). Both VLVHP and HVZP settings induced a downward shift of the PV curve at T2 h of the experimentation (Fig. 1).


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  		Fig. 1.   Static compliance curves of respiratory system. , Determination of pressure volume (PV) curve at time 0 (T0), representative of either ambient air- or hyperoxia-exposed rats (n = 6). Very low-volume high PEEP (; VLVHP; where PEEP is positive end-expiratory pressure) and high-volume zero PEEP (black-lozenge ; HVZP) are representative PV curves of rats either exposed to ambient air or hyperoxia and after 2 h of mechanical ventilation (MV; n = 6). Vt, tidal volume. Data are expressed as means ± SE.

Basically, a 2-h steady-state period of general anesthesia, either in SV or with MV, was able to increase overall alveolar-space protein content by a factor of at least five (Fig. 2). In addition, between MV subsets, VLVHP was the most distinctive by inducing a further twofold increase in protein content (P < 0.05 vs. T2 h SV). Hyperoxia (48 h) generally amplified total protein concentration in alveolar fluids regardless of spontaneous or mandated ventilatory conditions and regardless of screening (T0-T2 h; P < 0.05 vs. normoxia, except for MVLP; Fig. 2). VLVHP was again the ventilatory pattern inducing the highest modulation of protein content. By contrast and presumably because the majority of proteins in lavages originated from the bloodstream, CC-16 content in BALFs was decreased by MV (by a factor of 0.6-0.7), with the exception of MVLP (P < 0.05 vs. T2 h SV; Fig. 3A). Hyperoxia (48 h) decreased CC-16 content outright in BALFs before any type of MV (P < 0.05 vs. ambient air-exposed animals at T0), whereas a further drop in lung CC-16 was only observed with the VLVHP strategy after hyperoxia preexposure (P < 0.05 vs. T2 h SV; Fig. 3A).


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  		Fig. 2.   Total protein content in bronchoalveolar lavage fluids (BALFs). Protein concentration in alveolar fluids of experimental rats were measured at T0 (in a separate set of animals) and at the end of the steady-state period [time 2 h (T2 h)], as described in METHODS. Two populations of rats underwent several ventilatory strategies as follows: 1) control ambient air-exposed animals (filled bars) and 2) 48-h hyperoxia-exposed animals (open bars). Ventilatory strategies are indicated at bottom. The following two different patterns of ventilation were studied: spontaneous ventilation (SV) and MV. Four different types of MV were explored as detailed in Table 1: 1) VLVHP, 2) low-volume zero PEEP (LVZP), 3) medium-volume low PEEP (MVLP); and 4) HVZP. Data are expressed as means ± SD, with each group containing 6 rats. * Significant difference (P < 0.05) between hyperoxia-exposed and ambient air-exposed groups at T0. § Significant differences between T2 h and T0 groups in respective populations (P < 0.05). dagger  Significant difference (P < 0.05) at T2 h between rats mechanically ventilated and those spontaneously breathing.



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  		Fig. 3.   The 16-kDa Clara Cell (CC-16) contents in BALFs and serum. The same presentation as described in Fig. 2 is shown. Data are expressed as means ± SD, with each group containing 6 rats. A: CC-16 in BALFs. B: CC-16 in serum. * Significant difference (P < 0.05) between hyperoxia-exposed and ambient air-exposed populations at T0. § Significant differences (P < 0.05) between MV groups in respective populations and SV groups at T2 h.

In contrast with the above BALF data, CC-16 blood content was significantly and generally enhanced by a factor of >1.5-7 under positive-pressure MV (Fig. 3B), except for LVZP strategy (P < 0.05 vs. SV at T2 h). Hyperoxia (48 h) further accentuated this increase in blood CC-16 in all MV strategies, including LVZP, culminating in a 10-fold increase in the VLVHP group (P < 0.05 vs. T2 h SV; Fig. 3B). Analysis of the CC-16 serum-to-BALF ratio (a candidate marker of the transfer of pneumoproteins from lung to blood) and the T0-T2 h differential in CC-16 serum content (a time-related parameter of blood transfer) demonstrated that 1) MV is an important independent contributor of CC-16 blood content, 2) VLVHP and HVZP are the most inducing MV strategies of CC-16 transfer, and 3) proportionally, 48 h of hyperoxia further amplifies the pattern observed, with the exception of VLVHP where a tremendous disproportional elevation of CC-16 was noted (Fig. 4, A and B). Indeed, along with VLVHP MV, there was a 5.5- to 25-fold increase in the serum-to-BALF ratio and in the T0-T2 h differential of the CC-16 content, respectively (P < 0.05 vs. T2 h SV). In addition, and contrary to the alterations observed in BALFs, 48-h hyperoxia substantially increased CC-16 blood content outright (P < 0.05 vs. T0 SV; Fig. 4, A and B). Furthermore, there was a tight correlation between total protein content in BALFs and CC-16 serum-to-BALF ratios in all four groups of animals mechanically ventilated (n = 48, r2 = 0.402, P = 0.0001). Further increase of Vt to extreme levels (~30 ml/kg, VHVZP) exacerbated lung depletion and serum repletion of CC-16 compared with LVZP and HVZP groups (Fig. 5A). At 2 h, two out of six rats in this group were exhibiting similar CC-16 concentrations in both milieus (2.16 mg/l serum vs. 2.06 mg/l BALF and 2.33 mg/l serum vs. 2.4 mg/l BALF), suggesting the protein was freely crossing the A-C barrier. Macroscopic and microscopic evaluations confirmed diffuse, sometimes hemorrhagic, edema with hyaline membranes and vascular congestion (Fig. 5, B and C).


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  		Fig. 4.   Increase in serum-to-BALF (×10-3) CC-16 ratio (A) and enhanced differential of CC-16 content in serum with MV (B). Same presentation as described in Fig. 2 is shown. Data are expressed as means ± SD, with each group containing 6 rats. * Significant difference (P < 0.05) between hyperoxia-exposed and ambient air-exposed populations at T0. § Significant differences (P < 0.05) between MV groups in respective populations and SV groups at T2 h.



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  		Fig. 5.   Effect of incremental Vt with zero PEEP on rat lungs. A: progression of CC-16 BALF () and serum (black-lozenge ) concentrations with Vt increase: 7 ml/kg (VLVZP) and 19 ml/kg (HVZP; a) as described in Table 1 and 28.5 ml/kg (VHVZP, flow rate 11 ± 1 l/min; peak inspiratory pressure, 35 ± 3 cmH2O; respiratory rate, 20 ± 3 breaths/min; inspiration time, 0.5 ± 1 s; b). B: macroscopic view of dependent lung areas at the end of the MV period (a and b). C: representative microscopic view [optical microscopy, magnification ×400 (a) and ×200 (b), hematoxylin-eosin] of experimental lungs at the end of the MV period. Data in A are expressed as means ± SD, with each group containing 6 rats. Vt values (cc or ml/kg) on the x-axis are shown on a logarithmic scale.

Intratracheal instillation of KGF before the 48-h period of hyperoxia vs. ambient air conditioning allowed for a reduction in CC-16 blood leakage by 23.5 ± 1.2% (in the ambient air group) and 47 ± 3.9% (in the hyperoxia-exposed group) after 2 h of MV under one of the most leakage-inducing procedures (i.e., VLVHP). On the other hand, although the reduction in T0-T2 h CC-16 serum content was slight but significant with KGF treatment in ambient air (175 vs. 228%) and even greater after hyperoxia (307 vs. 410%; P < 0.05; Fig. 6A), the difference with KGF treatment for the serum-to-BALF ratio was present only after hyperoxia (P < 0.05; Fig. 6, A and B). Protein content in BALFs after KGF treatment returned to values similar to that observed with SV at T2 h in both ambient air- and hyperoxia-exposed groups (data not shown). Similar effects of KGF were observed with the HVZP group.


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  		Fig. 6.   Serum-to-BALF (×10-3) CC-16 ratio (A) and T0-T2 h differential of CC-16 content in serum with keratinocyte growth factor (KGF) pretreatment before VLVHP. Effect of KGF intratracheal instillation (5 mg/kg in 250 µl of vehicle) vs. vehicle intratracheal instillation alone (250 µl) on CC-16 lung and blood contents was studied in a subset of rats (n = 6/set) ventilated with VLVHP. Rats were initially submitted to a 48-h period of hyperoxia or ambient air, as described in METHODS. The same presentation as described in Fig. 2 is shown. Data are expressed as means ± SD. * Significant differences (P < 0.05) in KGF-treated and vehicle-treated groups at T2 h.

A decrease in arterial HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (a marker of metabolic acidosis appearance) was observed at T2 h of steady-state MV, with decreased concentrations in all conditions after hyperoxia. In ambient air-exposed lungs, this decrease was apparent only in the VLVHP group (P < 0.05 vs. SV; Fig. 7). Renal function was biochemically altered in all groups during the experimental period, as assessed by a 1.5- to 2-fold increase in blood creatinine (P < 0.05 vs. T0; Table 2). Of note, no significant difference in renal function was found between experimental groups at T2 h. MAP was 81.5 ± 4 mmHg at T0 in experimental groups and decreased overall to 63 ± 5 mmHg by the end of the 2-h period of observed ventilation, irrespective of hyperoxia or ambient air preexposure. VLVHP and HVZP groups exhibited further decreases of MAP at T2 h (56 ± 3 mmHg).


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  		Fig. 7.   T0-T2 h alterations in arterial blood HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> concentrations. The same presentation as described in Fig. 2 is shown. Data are expressed as differences between T0 and T2 h HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> concentrations in means ± SD, with each group containing 6 rats. * Significant differences between the MV and the SV groups in respective populations after a 2-h steady-state period of experimentation.


                              
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  		Table 2.   Blood creatinine content


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

MV is a determinant of lung protein transfer. A hallmark of mechanical VILI is pulmonary edema resulting from both increased filtration (by enhanced pulmonary intravascular pressure) and alteration of A-C membrane permeability (1, 17, 18). There is light and electronic microscopy evidence of endothelial and epithelial injury relative to MV (1, 17). Consecutive lung hyperpermeability has been studied for small solutes (i.e., 99mTcDTPA) and for larger solutes such as serum albumin and dextran (20, 38). Increased large protein content in distal air spaces lacks specificity in this experimental setup. Indeed, 2 h of supine SV under general anesthesia and all types of MV resulted in at least a 5- to 25-fold increase in total protein content in rat BALFs. The enhanced alveolar protein content arising from SV is of an unclear mechanism. Multiple factors, including dependent segmental atelectasis in a supine position, may have occurred, but there was no evidence of light microscopy lung injury or reactive alveolar cellularity (data not shown). By contrast, pneumoprotein CC-16 blood content was clearly more discriminative and was enhanced by positive-pressure MV compared with SV. Although CC-16 (protein and RNA) expression by distal epithelial cells has not been studied specifically, a trend toward a decrease in BALF content together with an increase of blood concentration suggests lung-to-capillary transfer with MV. This is exemplified further by CC-16 serum-to-BALF ratios and by the increment of T0-T2 h CC-16 blood content. Overdistended or overstretched lung air spaces, either by high end-inspiratory volumes with frequent cyclic reopening and no PEEP (HVZP) or by maintaining a very high PEEP with minimal flow rate cycling over the highest inflection point of the PV curve (VLVHP), are postulated propitious conditions favoring the transfer of CC-16 protein to the bloodstream, probably by potentiating the progressive increase in the pore radius, which is physiological at total lung capacity (12).

Lessons from the other experimental studies regarding the effects of MV to lung protein permeability are not easy to take in because of a huge variability in parameters. Indeed, extrapolations are sometimes problematic, depending on duration of MV, species types, or ventilatory parameters (whether the chest is open or not, whether the ex vivo lung is perfused or not, whether a pulmonary insult has been previously imposed or not, and, if so, whether the injury is endothelial, epithelial, or both). However, a "too high" PEEP (widely above the lower inflection point) generally leads to pulmonary edema with increased extravascular lung water, including epithelial lung fluid volume (8, 12, 42). Direct lung injury but also physiological disorders (such as the epithelial pump system dysfunction) are mechanistic in this way (27). This should remind us to keep in mind that very extreme PEEP (e.g., 24 cmH2O, or above, at 1.0 fraction of inspired 02 such as in the oxygenation goal chart of the ARDS network; see Ref. 6), even associated with a low "protective" ventilation, could be occasionally harmful. Indeed, no definitive MV rules should be drawn (24), whereas increasing PEEP in animals, even ventilated with low Vt, can cause edema (16). Comparable results of distal lung injury and A-C barrier permeability have also been described with zero end-expiratory pressure (ZEEP) (especially when combined with high-volume MV) or very low PEEP (below the lower inflection point) in study designs similar to our setup (8, 11, 27, 32).

Short-term hyperoxia-induced ALI is a sensitizer of MV-induced protein lung transfer, without evidence of synergistic effect (except for CC-16 blood content in the VLVHP group). Extensive injury of capillary endothelial cells and moderate injury to alveolar epithelial cells, together with interstitial edema, have been observed after 60 h of exposure to 100% oxygen in adult rats (10). Although there is no electron microspopic evidence of endothelial or epithelial cell alteration until 60 h of hyperoxia in adult rats, 48 h of hyperoxia substantially increased total protein content but decreased CC-16 content in BALFs (with a concomitant rise in blood concentrations) in our experiments. Consequently, it seems reasonable to postulate that some functional alteration of the A-C barrier occurs in hyperoxia-exposed lungs before any morphological changes and before all MV.

Other determinants that could have contributed to the rise of CC-16 blood content after MV (with or without preliminary hyperoxia). A potential problem using positive-pressure MV in rats arises from the depression of cardiovascular parameters with time (8, 16), resulting in a decreased blood pressure (MAP), and, by the way, to regional blood hypoperfusion, leading to metabolic acidosis highlighted by a decrease in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> arterial blood concentration (differential T0-T2 h). Indeed, metabolic acidosis with low pH, per se, has been suggested in favoring increased lung permeability and extravascular lung water in a model of septic shock by intravenous infusion of live Pseudomonas aeruginosa (37). In the present study design, only metabolic debt combined with hypercarbia allowed for arterial pH to approach ~7.2 in both VLVHP and LVZP. On the other hand, significant alterations in renal function occurring in conjunction with a reduction of the glomerular filtration rate, a main pathway for low-molecular-mass protein clearance (5, 22), most likely contributed to the increase in CC-16 blood content during the experimental period. Of note, in our study, medium-grade renal dysfunction was similar between experimental groups, whereas the alterations in CC-16 blood content were nonproportional and dissimilar (i.e., huge increase in CC-16 blood content in the hyperoxia/VLVHP group vs. a mild increase in CC-16 blood content in LVZP and MVLP not preexposed to hyperoxia, for similar creatinine levels). Mechanisms of renal dysfunction definitely point to systemic hypoperfusion and probably to a relatively conservative strategy of volume replacement to sampling and insensible loss.

Pneumoprotein transfer with ALI/hyperpermeability is the main source of CC-16 blood content. Ideally, an appealing marker of lung permeability would be of epithelial origin, sensitive and specific, and easy to measure in the bloodstream. This involves the recognition of the bidirectional nature of the alveolocapillary leakage of proteins recently emphasized by Hermans and Bernard (22). Lung protein leakage is usually described from blood to the air spaces in ALI (40). Yet, although pneumoproteins of epithelial origin can be detected in minute concentrations in the bloodstream under physiological conditions, they can also be found in severalfold increased amounts after ALI, when there is an enhanced passive diffusion through water-filled porous channels in the tight junctions (14, 15, 22, 30, 33, 34, 45). Actual candidates for pneumoprotein denominations are SP-A, SP-B, CC-16, and rTI40-HTI56 (14, 15, 22, 30, 33, 34, 45). The first three lung epithelioproteins exhibit relatively low molecular mass (~28-36, 8, and 16 kDa) and have recently been screened as biomarkers of hyperpermeability in patients with ALI/ARDS (14, 15, 22, 30) and in close relationship with the PaO2-to-FIO2 ratio and alveolar-arterial difference for PO2 (14, 15, 22, 30). No attempt has been made in the above studies to evaluate the contribution of MV adjustment to pneumoprotein leakage, as performed in this study. CC-16, a major secretory product of Clara cells in the terminal bronchioles of the lung, is one of the most abundant proteins in lung air spaces (~2% of total proteins) and exhibits immunosuppressive and anti-inflammatory properties (22). Clearance of CC-16 from the bloodstream is very swift (half-time <18 min) and is glomerular filtration rate dependent (14, 15, 22, 30). Thus, given the huge blood leakage observed in rats exposed to both hyperoxia and VLVHP MV, a subsequent temporary depletion of CC-16 should have occurred in the respiratory tract of these animals. Several A-C barrier permeabilizers, including epithelial and/or endothelial cell toxicants (e.g., lipopolysaccharide, Ipomeanol, O3, alpha -naphtylthiourea), have been reported to severely decrease CC-16 BALF content (2, 3, 23). Of note, matched elevations of CC-16 blood content were observed in the above experiments. In addition, these experiments reported a decrease in CC-16 mRNA expression in lung epithelial cells and a correlated lower number of protein-expressing cells (2, 3, 23). Although the above protein and RNA expressions were not specifically assessed in our study, these alterations may have occurred, together with a Clara cell dysfunction and deficient CC-16 release. Overall, both depressed CC-16 BALF and increased blood contents can account for several coexisting mechanisms in this work as follows: 1) decreased expression, production, and/or release at the alveolar interface (several inflammatory mediators may be contributors in this way) and 2) increased leakage and/or depressed clearance at the blood interface. However, these data likely suggest that enhanced CC-16 blood content is a marker of airspace-to-capillary leakage, and this can even lead to "both side" leveled concentrations of CC-16 in extreme conditions (e.g., VHVZP).

Prophylactic reinforcement of the epithelial barrier with KGF reduces CC-16 leakage. KGF is a powerful repair factor for lung epithelial cells (35). Systemic or local intratracheal instillation of this cytokine induces distal air space epithelial cell proliferation over several days and can help lung restitution when instilled before bleomycin-, radiation-, acid-, and oxygen-induced ALI (4, 45). Additional potential modes of action in KGF-induced regulation of alveolar epithelial barrier are also suggested, such as a reduction in cell apoptosis, increased expression of surfactant-associated proteins, and upregulation of alveolar liquid clearance (4, 45). Indeed, VILI produces lung disability to clear edema, dysfunction of surfactant, and, of course, alteration of the epithelial side of the A-C barrier (17, 26, 27). Prolonged hyperoxia produces similar effects but does not change or increases the ability of the lung to clear edema (19). Recently, the preventing capacity of KGF to VILI has been reported using an ex vivo model specific in several points (46) as follows: KGF was systemically administered, only large-molecular-mass protein transfer from perfusate to air spaces was measured, and volume overdistension was moderate in the aggressive arm (i.e., Vt ~4 ml and ZEEP). Reduction in lung water content and dextran BALF concentrations (46), together with decreases in both CC-16 capillary leakage and total protein content in BALFs (in our study), were severalfold with KGF treatment. Although the latter data suggest that KGF controls blood-to-lung protein transfer more efficiently than the transfer from lung to blood, they also further the argument for an epithelial dysfunction as a major determinant of lung-to-blood transfer, whereas KGF was instilled intratracheally. On the other hand, KGF is already known to decrease CC-16 mRNA expression in the lung (41). The latter effect likely contributed in lowering CC-16 BALF content but also in leveling out variations in serum-to-BALFs ratios and T0-T2 h CC-16 blood concentrations.

In summary, positive-pressure MV alters A-C barrier permeability, even in the normal lung. CC-16 shows tremendous leakage with lung overdistension generated by high PEEP or high volume. Preliminary hyperoxia sensitizes CC-16 transfer to the bloodstream. Additional determinants of CC-16 blood transfer are systemic hypoperfusion and accompanying renal dysfunction. KGF is an effective molecule in reducing combined MV- and hyperoxia-induced lung hyperpermeability to CC-16.


    ACKNOWLEDGEMENTS

This work was supported by the Clinical Research Center of Sherbrooke, the Association Pulmonaire du Québec. O. Lesur is a research scholar of the Fonds de Recherche en Santé du Québec.


    FOOTNOTES

Address for reprint requests and other correspondence: O. Lesur, Groupe de Recherche en Physiopathologie Respiratoire, Centre de Recherche Clinique, CHU Sherbrooke, Qc Canada, J1H 5N4 (E-mail: Olivier.Lesur{at}USherbrooke.ca).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published October 25, 2002;10.1152/ajplung.00384.2001

Received 28 September 2001; accepted in final form 21 October 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Am J Physiol Lung Cell Mol Physiol 284(2):L410-L419
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