INTRODUCTION

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Mechanical ventilation of critically ill patients is frequently complicated by adverse events including nosocomial pneumonia, hemodynamic instability, and pulmonary barotrauma (1). A number of investigators have shown physiologic and morphologic changes in the lung caused by mechanical ventilation and characterized by increased permeability edema and diffuse alveolar damage (2, 3). Recently, much attention has been focused on the role of ventilator-induced lung injury (VILI) in influencing cytokine release and the clinical outcome of the acute respiratory distress syndrome (ARDS) (4, 5).

Initially, high inflation pressures were thought to be resonsible for the lung damage observed in VILI, but subsequent investigations have found that large tidal volumes rather than high inflation pressures are responsible for this (2, 6). High tidal volumes overdistend alveoli, resulting in disruption of the integrity of the alveolar epithelial layer and, secondarily, of alveolar-capillary endothelial cell junctions (7). The resulting alveolar-capillary damage allows exudation of intravascular water and proteins into the interstitium and alveolar spaces (2).

Several protective strategies against lung injury from mechanical ventilation have been investigated in both animal experiments and clinical trials. Considerable attention has been directed toward reduction of tidal volume (VT) as a means of avoiding ventilator-associated lung injury (VALI) (2, 3, 8). The recently completed National Institutes of Health ARDS Network trial found less mortality associated with the use of smaller tidal volumes (9). A previous clinical trial suggested that the use of positive end-expiratory pressure (PEEP) based on the flow-volume curve might be beneficial (10).

Keratinocyte growth factor (KGF) is a member of the fibroblast growth factor (FGF) family that was purified and characterized in 1989 (11). This heparin-binding mitogen is secreted primarily by fibroblasts, and targets epithelial cells. Both in vitro and in vivo studies have shown hyperplasia caused by overgrowth of type II pneumocytes in response to KGF, peaking approximately 72 h after administration of this factor (12, 13).

KGF has been shown to reduce lung injury caused by a variety of agents, including -naphthylthiourea, hyperoxia, hydrogen peroxide, and acid instillation (13). Therapies that prevent or reduce VILI may have a significant impact on patient outcome in both the intensive care unit (ICU) and over the long term. We investigated the effect of pretreatment with KGF on the development of VILI in an isolated, perfused rat lung model, and examined a potential mechanism of its action.

	METHODS

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Animals

Male Sprague-Dawley specific pathogen-free rats (body weight 250 to 274 g) were obtained from Charles River Breeding Laboratories (Wilmington, MA) and housed in the Louisiana State University Medical Center (LSUMC) Animal Care Facility. Animals were allowed food and water ad libitum. The experimental protocol was approved by the LSUMC Institutional Animal Care and Use Committee.

Intravenous Injections

Intravenous injection of KGF (1 mg/kg in 1 ml PBS) (generously provided by Thomas R. Ulich, M.D., of Amgen, Inc., Thousand Oaks, CA) or phosphate-buffered saline (PBS) daily for 3 d was accomplished via the dorsal penile vein with a 30-gauge needle after induction of anesthesia by metofane inhalation.

Isolated Perfused Lung Preparation

The isolated perfused lung model has been previously described (14, 17). Briefly, animals were anesthetized with an intramuscular injection of ketamine and xylazine (50 mg/kg + 5 mg/kg, respectively; Fort Dodge Laboratories, Fort Dodge, IA), and the trachea was cannulated after anesthesia was achieved. The lungs were ventilated with a constant VT of 7 ml/kg of 95% oxygen and 5% carbon dioxide at 60 breaths/min, with 5 cm H₂O PEEP provided by a small-animal ventilator (Harvard Apparatus, South Natick, MA). A catheter was inserted into the left carotid artery, and after the administration of 1.0 ml of sodium heparin solution (5,000 U/ml) the animal was exsanguinated. The chest was then opened and the main pulmonary artery was exposed. The pulmonary artery was cannulated through a right ventriculotomy and the lungs were perfused with Krebs-Henseleit buffer. Next, the left atrium was cannulated via a left ventriculotomy, and a device allowing application of variable resistance was placed on the left atrial cannula. The lungs and heart were removed en bloc from the chest and placed in a heated water jacket. The lungs were perfused with a mixture of one part Krebs-Henseleit buffer to three parts heparinized whole blood at a rate of 4.5 ml/min with a Masterflex pump (Cole-Parmer Instrument Co., Barrington, IL). The perfusate was circulated through a 37° C heat exchanger. The perfusate reservoir was constantly stirred with a magnetically driven stirrer bar to provide adequate mixing. Airway pressure (Paw), pulmonary artery pressure (Ppa), left atrial pressure (Pla), and the pressure in the perfusate reservoir were recorded continuously with an eight-channel recorder (Grass Instrument Co., Quincy, MA). After an initial 15-min period of stabilization, Pla was raised to 3 mm Hg. Fluid draining via the left atrial catheter was returned to the perfusate reservoir. Another reservoir was positioned below the isolated lungs and was used to collect fluid that effluxed from the surface of the lungs (lymph flow). To quantify lung flux, we correlated changes in the perfusate reservoir pressure, which were recorded continuously during the experiment, with volume changes in the perfusate reservoir, as well as with lymph flow. Lung water was calculated as the difference between lung flux (the volume lost from the perfusate reservoir) and lymph flow (the volume effluxing from the surface of the lungs). Dynamic compliance (Cdyn) was calculated at the beginning and end of the ventilatory period as the quotient of the VT and the Paw.

Wet-to-Dry Lung Weight Ratios

In order to confirm that lung flux correlated with the volume change in the perfusate reservoir, wet-to-dry (W:D) lung weight ratios were calculated at the end of the experiments. The lungs were removed and the wet weight was recorded. The lungs were then placed in an incubator at 37° C for 7 d, at the end of which time the dry weight was recorded and the W:D weight ratio was calculated.

Experimental Protocol

The isolated lungs were ventilated and perfused ex vivo for a total of 60 min after the initial stabilization period. Three experimental groups were studied: (1) a control group that was ventilated with a noninjurous strategy (VT 8 ml/kg, PEEP = 5 cm H₂O, and respiratory rate = 60 breaths/min); (2) a VILI group that was pretreated as described earlier with PBS and ventilated with a VT of 17 ml/kg, zero end-expiratory pressure (ZEEP), and a respiratory rate of 60 breaths/ min; (3) a KGF group that was pretreated with KGF and ventilated with the same strategy as the VILI Group.

Alveolar-Space Protein

At the conclusion of the ventilatory period, the lungs were removed from the holding chamber and lavaged with 30 ml of Dulbecco's PBS containing 3 mM ethylenediaminetetraacetic acid (EDTA) at room temperature (21° C) in 5-ml aliquots. The fluid was centrifuged at 200 × g for 10 min, and the supernatant was stored at 80° C. A total protein assay was performed with the bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL). A standard curve was prepared from known amounts of albumin to calculate the values of protein recovered in the bronchoalveolar lavage fluid (BALF). Total protein was calculated as the product of the concentration of protein and the amount of BALF recovered.

Effect of KGF on Alveolar Capillary Barrier Permeability During VILI

In a separate set of experiments, 0.5 ml of a 0.5 mg/ml solution of fluorescein isothiocyanate (FITC)-labeled dextran (FITC-dextran) (73,000 kD) was added to the lung perfusate fluid after the initial 15-min stabilization period. Bronchoalveolar lavage (BAL) was performed as described earlier, and the fluorescence of the supernatant was measured spectrophotometrically (Model 650-40; PerkinElmer, Inc., Norwalk, CT) with an excitation wavelength of 495 nm and emission wavelength of 517 nm. A standard curve was prepared from known amounts of FITC-dextran to calculate the values recovered in the BALF. Total FITC-dextran was calculated as the product of the concentration and the amount of BALF recovered. The total FITC- dextran was divided by the lung wet weight to obtain a value of FITC- dextran per gram of tissue. This value normalizes the amount of recovered FITC-dextran for total lung water.

Increased total FITC-dextran in BALF indicates an increase in blood-gas barrier permeability, since the large FITC-dextran molecule normally does not transgress an intact capillary-alveolar barrier. Decreased alveolar accumulation in the KGF group would indicate a protective effect of KGF on barrier permeability (18).

Histology

Histologic sections were obtained after intravenous administration of KGF as described earlier, and of intravenous PBS as a negative control. Positive controls were procured by collecting specimens 72 h after intratracheal administration of KGF (5 mg/ml in 0.5 ml PBS). After killing by transection of the inferior vena cava, each animal's lungs were excised en bloc, and were then inflated with 10% formaldehyde at 15 cm H₂O pressure and stored in formaldehyde-filled containers. The lungs were then embedded in paraffin and sagittal sections were cut. Representative histologic samples were stained with hematoxylin and eosin (H&E). Tissue sections were examined at a magnification of ×400 and photomicrographs of the sections were made.

Statistical Analysis

Group mean data were compared through analysis of variance to detect differences between groups, and through unpaired t tests for differences between two groups. Where appropriate, a Bonferroni-Dunn multiple comparison test was performed. A value of p  0.05 was accepted as statistically significant. All data are presented as mean ± SEM.

	RESULTS

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Gravimetry

The W:D lung weight ratios in the VILI group (n = 11) were significantly higher than those in the control (n = 8) group (10.81 ± 0.96 versus 5.26 ± 0.15 [mean ± SEM]; p < 0.05). The KGF group (n = 14) had significantly reduced W:D ratios for lungs subjected to an injurious ventilatory strategy (KGF: 6.54 ± 0.68 versus VILI: 10.81 ± 0.96; p < 0.05), which were not statistically different from those for the control group ventilated with a noninjurious strategy (KGF: 6.54 ± 0.68 versus control: 5.26 ± 0.15; p = 0.32). These results are illustrated in Figure 1.

View larger version (34K): [in this window] [in a new window]   Figure 1.   W:D lung weight ratios. *p < 0.05 versus control; +p < 0.05 versus VILI.

Lung Water

The calculated increase in lung water is shown in Figure 2. There was a minimal increase (0.06 ± 0.74 ml) in lung water in the control group. The VILI group had a significant gain in lung water (3.35 ± 0.37 ml; p < 0.05) as compared with the control group. The water gain in the KGF group (0.40 ± 0.31 ml) was significantly less than in the VILI group (p < 0.05), and was not statistically different from that in the control group (p = 0.63).

View larger version (24K): [in this window] [in a new window]   Figure 2.   Changes in lung water (ml). *p < 0.05 versus control; +p < 0.05 versus VILI.

Compliance

The lungs of the control group had a greater Cdyn than those of either the VILI or the KGF group (p < 0.05) at the beginning of the experiment, which was most likely due to ventilation on a different segment of the pressure-volume curve. There was no difference in initial Cdyn between the VILI and KGF groups (p = 0.73). The change in Cdyn over the course of the experiment was significantly greater in the VILI group (0.099 ± 0.008 ml/mm Hg) than in the KGF (0.056 ± 0.009 ml/mm Hg; p < 0.05) or the control group (0.0004 ± 0.018 ml/mm Hg; p < 0.05) (Figures 3A and 3B).

View larger version (34K): [in this window] [in a new window]   View larger version (28K): [in this window] [in a new window]   Figure 3.   (A) Initial Cdyn (ml/mm Hg). *p < 0.05 versus control. (B) Change in Cdyn (ml/mm Hg). *p < 0.05 versus control; +p < 0.05 versus VILI.

Alveolar-Space Protein

The protein concentration in BALF was significantly higher in the VILI group (0.82 ± 0.11 µg/ml) than in either the control group (0.26 ± 0.03 µg/ml; p < 0.05) or the KGF group (0.33 ± 0.11 µg/ml; p < 0.05). There was no statistical difference between the KGF and control groups (p = 0.69). These relationships held true when the total alveolar-space protein content was calculated (Figures 4A and 4B).

View larger version (38K): [in this window] [in a new window]   View larger version (32K): [in this window] [in a new window]   Figure 4.   (A) BALF protein concentration (µg/ml). *p < 0.05 versus control. +p < 0.05 versus VILI. (B) Total alveolar space protein (µg). *p < 0.05 versus control; +p < 0.05 versus VILI.

FITC-Dextran Measurements

The BALF concentration of FITC-dextran in the VILI group (0.27 ± 0.05 µg/ml) was greater than in both the control (0.06 ± 0.02 µg/ml; p < 0.05) and the KGF (0.06 ± 0.03 µg/ml; p < 0.05) groups (n = 5 in all groups). There was no difference between the control and KGF groups (p = 0.97) in BALF FITC- dextran. Both total alveolar-space FITC-dextran and the ratio of FITC-dextran to lung dry weight were significantly greater in the VILI group than in either the control group or the KGF group (Figures 5A to 5C).

View larger version (29K): [in this window] [in a new window]   View larger version (30K): [in this window] [in a new window]   View larger version (33K): [in this window] [in a new window]   Figure 5.   (A) BALF FITC-dextran concentration (µg/ml). *p < 0.05 versus control; +p < 0.05 versus VILI. (B) Total alveolar space FITC- dextran (µg). *p < 0.05 versus control; +p < 0.05 versus VILI. (C) Total FITC-dextran to wet lung weight ratios (µg/g). *p < 0.05 versus control; +p < 0.05 versus VILI.

Histology

Lung sections from animals pretreated with intravenous KGF, intravenous PBS, and intratracheal KGF were stained with H&E and examined by light microscopy at ×400 lt magnification. Sections from animals pretreated with intravenous KGF showed diffuse, mild hyperplasia of alveolar type II pneumocytes (Figure 6C). The sections from the PBS-pretreated animals revealed normal rat alveolar architecture (Figure 6A), whereas sections from the positive controls given intratracheal KGF demonstrated marked, slightly inhomogeneous hyperplasia of alveolar type II pneumocytes (Figure 6B).

View larger version (104K): [in this window] [in a new window]   View larger version (126K): [in this window] [in a new window]   View larger version (108K): [in this window] [in a new window]   Figure 6.   (A) Normal rat lung histologic section after intravenous administration of PBS, stained with H&E. (Original magnification: ×400.) (B) Marked alveolar type II cell hyperplasia is seen after intratracheal administration of KGF in H&E-stained rat lung histologic sections. (Original magnification: ×400.) (C) Mild, diffuse alveolar type II pneumocyte hyperplasia in H&E-stained rat lung histologic sections after intravenous administration of KGF. (Original magnification: ×400.)

	DISCUSSION

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The study showed that pretreatment with KGF attenuates pulmonary injury in an isolated, perfused rat lung model of VILI. The ventilator-induced lung damage was produced by ventilation with both a relatively large VT and ZEEP. The reduction in lung injury with KGF was supported by a decrease in W:D lung weight measurements, less accumulation of lung water, and maintenance of Cdyn. The alveolar-space protein and BALF FITC-dextran measurements further supported mediation of the effect of KGF, at least in part, by preservation of the integrity of the alveolar-capillary barrier.

It is unclear whether the type II pneumocyte hyperplasia induced by KGF has a direct effect in preventing VILI. Even with the extensive hyperplasia seen with intrathecal KGF, most of the alveolus is covered by type I pneumocytes. It may be that KGF indirectly induces changes in the alveolar capillary membrane.

We and others have shown in other models of lung injury that KGF exerts its protective effect via an upregulation of alveolar liquid clearance (17, 19, 20). This does not appear to be the predominant protective mechanism in the rat lung model of VILI used in the present study. The KGF group did not exhibit an increase in either alveolar protein or FITC-dextran, which would be expected if capillary leak was present but accelerated fluid clearance was the dominant protective mechanism. Thus, maintenance of integrity of the alveolar-capillary barrier is an important factor in the mechanism by which KGF prevents VILI.

The paracrine mitogenic effects of exogenous KGF on the epithelium peak after 2 to 3 d, and appear morphologically similar to the reactive type II pneumocyte hyperplasia observed in humans in the setting of acute lung injury (13). Borok and fellow investigators demonstrated that KGF inhibited transdifferentiation of monolayers of cultured alveolar type II cells to a type I phenotype for up to 8 d (21). This may allow enhanced metabolic function of the type II cells and may accentuate any structural changes associated with hyperplasia. In vivo, alveolar histology returns to normal after 7 d of KGF administration to rats (13), suggesting that the pneumocytes become resistant to KGF, allowing repopulation of the alveolus with type I cells.

Both intratracheal and intravenous administration of KGF induce type II pneumocyte hyperplasia (13, 22). Although the response in our study was more dramatic with intratracheal than with intravenous administration, the hyperplasia observed after intratracheal KGF tended to be more focally present in the lung, which is most likely reflective of inhomogeneous distribution of the growth factor (13). Most studies of the protective effects of KGF have utilized an intratracheal approach to its administration, but intravenous KGF was recently shown to protect against bleomycin- and hyperoxia-induced lung injury and mortality (22). Intravenous administration of KGF leads to more diffuse proliferative changes, though the hyperplasia is less pronounced than that seen after intratracheal administration (13). A systemic mode of delivery was used in our model of VILI.

Although KGF leads to a number of physiologic changes in the alveolar epithelium, which have been suggested to protect the lungs, in vitro evidence supports a protective effect of KGF via attenuation of increases in permeability. Recently, both pre- and posttreatment with KGF were shown to decrease both hydrogen peroxide- and radiation-induced increases in permeability in a human airway epithelial cell line as determined by FITC-albumin flux across cell monolayers (15, 23). Our results extend this concept to an ex vivo model of lung injury. Adding a fluorescein-labeled, high-molecular- weight dextran to lung perfusate solution, and then determining the quantity of dextran in the alveolar space (as assessed by BAL), allowed an estimation of microvascular permeability. The use of this method to estimate leak into the alveolar space is comparable to the use of techniques involving radiolabeled tracers, and has recently been validated in our laboratory as a means of assessing loss of integrity of the alveolar epithelial barrier (18).

KGF may affect alveolar-microvascular permeability in a number of ways. The well-documented type II pneumocyte hyperplasia suggests a direct effect on epithelial cells. It has been suggested that KGF may reduce fluid flux across the alveolar epithelium by stabilizing the pneumocyte cytoskeleton. Treatment with KGF was found to increase staining of actin filaments and to reduce cytoskeletal disruption after hydrogen peroxide injury in airway cell monolayers (15). A preliminary study by Margulies and coworkers examined the effect of KGF on cyclic mechanical stretch in cultured alveolar type II pneumocytes. KGF reduced cell death in stretched cells, but its effect was not significantly different from what was seen in controls when cells were not stretched (24). Because VILI probably involves overdistention of alveolar units and mechanical disruption of the epithelial membrane, one possibility for the mechanism by which KGF preserves barrier function may be mediated by a reduction in strain-induced cell death, thereby preserving the alveolar epithelium.

It is possible that indirect effects of KGF affect alveolar-capillary permeability. In skin and other nonpulmonary tissues, KGF has altered collagen deposition and/or the production of various matrix metalloproteinases. This may account for the described protective effect of KGF despite limited histologic changes (16, 25).

Cell-cell and cell-extracellular matrix interactions may influence permeability characteristics of the epithelial layer. Preliminary reports indicate that FGF upregulates expression of integrin-1, a subunit of integrin-51, which is proposed to be the primary adhesion molecule of alveolar type II pneumocytes. Moreover, in association with its enhancement of integrin-1 expression, FGF increased attachment of alveolar type II pneumocytes to fibronectin, a basement membrane component (30).

A further point is that, although most of the impedance to protein leakage into the alveolar space is due to epithelial integrity, the pulmonary microvasculature may be integral to the protective mechanism of KGF (31). This growth factor can promote endothelial proliferation indirectly, through the induction of vascular endothelial growth factor (32). Moreover, KGF has recently been shown to induce neovascularization in the eye, and to maintain adrenal capillary endothelial cell barrier integrity in vitro (33).

In addition to changes to the alveolar-capillary barrier, attenuation by KGF of the permeability defect in VILI might be due to limitation of the severity of injury through other mechanisms. Thus, KGF induces alveolar sodium export and fluid clearance (17, 20). Alveolar flooding has been shown to exacerbate VILI (34), suggesting that the accelerated alveolar liquid clearance observed with KGF could reduce air space fluid, thus ameliorating injury. Likewise, KGF increases surfactant production, which might indirectly prevent a permeability defect (35). Since VILI is more pronounced in surfactant-depleted models (38), and VILI itself adversely affects surfactant aggregate conversion (39), this increase in surfactant production may be a potential mechanism of action of KGF.

Despite recent clinical advances in limiting VALI through reduction in VT and the use of PEEP, our knowledge of such injury remains incomplete. These measures may only partly reduce the detrimental effects of mechanical ventilation. Changes in ventilatory strategy can also have limiting negative effects, such as impairment of cardiac output with high levels of PEEP, and difficulty in maintaining oxygenation with lower tidal volumes (9, 40). Thus, a pharmacologic intervention that attenuates VALI, either alone or in combination with these other measures, could prove a useful clinical tool.

In conclusion, we have demonstrated a protective effect of KGF pretreatment in an ex vivo rat model of VILI. The protective effect appears to be mediated by a mechanism that preserves the alveolar-capillary barrier. Although recent studies investigating reduction in VT have documented a reduction in lung injury and mortality with this technique, difficulty in maintaining adequate gas exchange and acid-base balance indicates that alternative approaches may be useful.