Halothane

Study objectives: Fibroblast growth factor (FGF)-2 is one of the most powerful angiogenic growth factors to be evaluated as an agent for the promotion of angiogenesis. The aim of this study is to investigate whether intratracheal administration of controlled-release FGF-2 microspheres restores pulmonary function in beagle dogs with emphysema.

Design: Randomized, controlled, experimental animal study.

Subjects: Eighteen Wister rats and 15 adult beagle dogs.

Methods: In the rat study, we compared the time profiles of the radioactivity remaining after intratracheal injection of [sup.125]I-labeled FGF-2, either incorporated with the controlled-release microspheres or as an aqueous solution. In the dog study, elastase-induced emphysema models were developed in 10 animals, classified into the following three groups: control group (n = 5), emphysema model with empty microspheres-treated group (FGF - group, n = 5), and emphysema model with FGF-2 containing microspheres-treated group (FGF + group, n = 5).

Results: In the rat study, controlled-release microspheres maintained higher whole-lung FGF-2 concentrations after intratracheal administration. In the dog study, Pa[O.sub.2] in the FGF + group was significantly higher than in the FGF - group after treatment. Pulmonary perfusion dynamic MRI revealed significant improvement in the signal intensity of damaged lung with the FGF + group. Linear intercept of the FGF + group was significantly reduced than the FGF - group.

Conclusion: Results indicate that intratracheal administration of FGF-2 induced an increase in pulmonary blood flow in the damaged lung and led to recovery of pulmonary function. The controlled-release microsphere system increased the effectiveness of FGF-2.

Key words: angiogenesis; COPD; emphysema; fibroblast growth factor; regeneration

Abbreviations: FGF = fibroblast growth factor; FGF - group = emphysema model with empty microspheres-treated group; FGF + group = emphysema model with FGF-2 containing microspheres-treated group; Lm = mean linear intercept; P/V = pressure/volume; LVRS = lung volume reduction surgery

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Lung volume reduction surgery (LVRS) improves lung function, exercise capacity, and quality of life in patients with advanced emphysema by allowing the remaining pulmonary parenchyma and the respiratory muscles to function more effectively. Although the physiologic and symptomatic benefits of LVRS have, on average, been impressive, the substantial rates of morbidity and mortality associated with major thoracic surgery and general anesthesia in an elderly, debilitated population have limited the clinical utility of the procedure. Moreover, patients with the most advanced disease have higher surgical mortality, suggesting that LVRS is not suitable for those with severe disease). (1,2) There is currently no therapy for the treatment of lung emphysema. Noninvasive treatment is desirable for severe emphysema patients.

Fibroblast growth factor (FGF)-2 is one of the most powerful angiogenic growth factors to be evaluated as an agent for the promotion of angiogenesis. Patients with severe emphysema have higher-than-normal pulmonary arterial pressure. Severe emphysema also tends to produce diffuse microvessel abnormalities in the pulmonary peripheral arteries. (3) Induction of a collateral pulmonary vessel network is a potent method of providing effective relief from dyspnea, general fatigue, and other symptoms in emphysema. Polymer hydrogels composed of gelatin have previously been demonstrated to be suitable matrices for the controlled release of growth factors because of their biosafety and the fact that they are highly inert toward protein drugs. (4) Biodegradable gelatin microspheres incorporating FGF-2 have been developed using acidic gelatin hydrogels. The use of these microspheres enables FGF-2 to be released at the site of action over a sufficiently long period of time to act effectively, in remarkable contrast to free FGF-2. (5) The aim of the present study was to investigate whether there is a benefit of bronchoscopic administration of FGF-2 on elastase-induced emphysema animals.

MATERIALS AND METHODS

Distribution of FGF-2 Intratracheal Administration in Rats

Eighteen female Wister rats weighing 250 to 300 g and 15 adult beagle clogs weighing 10.0 to 14.9 kg were used in this study. The study protocol was approved by the Kyoto University Ethics Committee for Animal Research. All animals received humane care in compliance with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources, National Research Council, and published by the National Academy Press.

The rats were anesthetized with pentobarbital sodium, and the trachea was exposed. All the FGF-2 was labeled by [sup.125]I. [sup.125]I-labeled FGF-2 was injected via 0.3 mL of solution to the trachea (FGF solution group, n = 9). [sup.125]I-labeled-FGF-2 microspheres were injected in 0.3 mL of suspended solution to the trachea (FGF microsphere group, n = 9). Lung tissues were obtained at different time intervals: 24 h, 72 h, and 7 days. The radioactivity remaining was calculated from the wbole lung on a gamma counter (ARC-301B; Aloka; Tokyo, Japan).

Preparation of the Elastase-Induced Emphysema Model

The 15 beagle dogs were classified at random into the following three groups: control group, emphysema model with empty microspheres-treated group (FGF- group), and emphysema model with FGF-2 containing microspheres-treated group (FGF +) group. Models of elastase-induced emphysema were developed in the FGF - and FGF + groups. All interventions and physiologic measurements were performed under general anesthesia with ketamine hydrochloride (10 mg/kg) and xylazine (30 mg/kg), and mechanical ventilation was administered through an endotracheal tube. A 9.0-mm diameter endotracheal tube was inserted into the trachea under bronchoscopic guidance and attached to a mechanical ventilator. Continuous monitoring included ECG, oxygen saturation by reflectance oximetry using a sensor clipped to the ear, and body temperature by means of a rectal probe. A bronchoscope (5 mm outside diameter and 60 cm working length) was introduced through the indwelling endotracheal tube and advanced to the left segmental bronchus. Then, 40 mg (3,000 U) of porcine pancreatic elastase (Nakarai Tesque; Kyoto, Japan) was dissolved in 5 mL of saline solution and sprayed into all segmental bronchi of the left lung through the instrument channel of the bronchoscope using a spray infusion catheter (Olympus Optical; Tokyo, Japan) in the elastase-induced emphysema models. Each elastase dose was divided into 10 portions, each of which was sprayed in a different area to make a model of diffuse emphysema at the level of the left segmental bronchus. The right lung was preserved intact as a control.

Arterial Blood Gas and Pressure/Volume Relationships

Assessment of pulmonary function and MRI were performed before elastase administration (baseline), 4 weeks after elastase administration, and 4 weeks after treatment. For assessment of pulmonary function, dogs were anesthetized, intubated, and maintained on < 3.0% halothane. Arterial blood pH, PaC[O.sub.2], Pa[O.sub.2], and percentage of oxygen saturation were measured. Mechanical ventilation was set at a breathing frequency of 10 breaths/min, the inspiratory time was set to 33% of the breathing period, and the fraction of inspired oxygen was 0.2. Tidal volume was set to 18 mL/kg. Arterial blood gas samples were obtained from the right femoral artery 15 min after mechanical ventilation was started. A blood gas and acid-base analyzer (ABL-620; Radiometer; Copenhagen, Denmark) was used for measurements. Pressure/volume (P/V) relationships and expiratory capacity were also measured under general anesthesia with intubation. The intratracheal cavity was inflated to various pressures (5 to 70 cm [H.sub.2]O), and the endotracheal tube was then clamped tightly. A plethysmograph (HI-701; Nihon Kohden; Tokyo, Japan) was connected to the endotracheal tube and the clamp was released, and then the expiratory capacity was measured. The expiratory capacity when the intratracheal cavity was inflated to a pressure of 40 cm [H.sub.2]O (expiratory capacity, 40 cm [H.sub.2]O) was used for calculations.

Dynamic Contrast-Enhanced MRI

All MRI studies were performed (1.5 T Sonata; Siemens Medical Systems; Erlangen, Germany) with a maximum amplitude of 40 mT/m and a rise time of 0.6 ms, using a phased-array body coil with four active segments. A turbo fast low-angle shot sequence optimized for projection imaging was used for dynamic contrast-enhanced MRI. (6) The following image parameters were used: echo time/repetition time, 1.3.5/350 ms; flip angle, 8[degrees]; readout bandwidth, 500 Hz/pixel; section thickness, 20 mm; field of view, 300 to 350 x 140 to 170 mm; image matrix, 110 x 256; and voxel size, 1.3 x 1.2 x 20.0 [mm.sup.3]. For the MRI scan, each dog was anesthetized and a 16-gauge IV catheter was introduced into the right internal jugular vein. The dog was then fixed in a supine position and 3 mL of gadopentetate dimeglumine (Magnevist; Nihon Sobering; Osaka, Japan) was administered as an IV bolus over 1 s. The contrast agent was administered immediately after the start of the dynamic imaging procedure. A total of 170 axial images were acquired to provide consecutive measurements over the 60-s scan time. The image immediately before the image showing any vascular enhancement was utilized as a mask image for subsequent image subtraction. For the imaging procedure, the signal intensity curves were measured from the right and left lung parenchymal areas (plotting area) separately. The mean signal intensity was calculated from the signal intensity curve during the 60-s scan time. The flow-volume ratio was calculated from the mean signal intensity of the left lung to the right lung, according to the following equation:

Flow-volume ratio = mean signal intensity in left lung/ mean signal intensity in right lung

Administration of FGF-2 Microspheres

Human recombinant FGF-2 was supplied by Kaken Pharmaceutical Company, Tokyo, Japan. Gelatin was isolated from bovine bone collagen by an alkaline process using CaO[H.sub.2] (Nitta Gelatin Company; Osaka, Japan). FGF-2 microspheres with a diameter of approximately 10 [micro]m were prepared as described previously by glutaraldehyde cross-linking of gelatin. (4,5) After washing with acetone (4[degrees]C), the microspheres were recovered by centrifugation. FGF-2 was radioiodinated and incorporated into the microspheres over a 1-h period before use. In the FGF + group, a total of 200 [micro]g of FGF-2 was incorporated into each 4.0 mg of gelatin hydrogel microspheres. After performing a bronchoscopic examination, 4.0 mg of FGF-2 microspheres were suspended in 5 mL of saline solution and sprayed into the emphysematous left lung using a spray infusion catheter in 10 divided doses in a different area. The FGF - group were sprayed with 4.0 mg of gelatin hydrogel microspheres without FGF-2 using the same procedure into the left lung.

Histologic Measurement

Four weeks after treatment, the dogs in each group were euthanized by injection of pentobarbital sodium. The lung tissues and heart, along with the trachea, were resected en bloc. The heart and mediastinal tissues were removed, and the lungs with the attached trachea were weighed. The lungs were immediately inflated with 10% neutral buffered formalin solution via a tracheal cannula at a pressure of 25 cm [H.sub.2]O until the pleura became tense. The trachea was then ligated, and the lungs were fixed further by immersion in formalin solution for 48 h. The inflated lung volume was measured by the water replacement method, and the left and right lungs were measured separately. (7) Twenty 2 x 2 x 2-cm blocks were randomly cut from the whole area of the lung per animal. The blocks were then embedded in paraffin before being cut into 3-[micro]m-thick sections and stained with hematoxylin-eosin. All the sections were used to measure mean linear intercept (Lm), a commonly used stereologic indicator of alveolar airspace enlargement in emphysema, which was calculated as described. (8) Sixty fields per animal at 40 x magnification were chosen at random to measure Lm.

Statistical Analysis

Data are expressed as mean (SD). Data were analyzed by analysis of variance using statistical software (StatView for Windows, version 5.0; SAS Institute; Cary, NC). Differences between groups were identified by a Scheffe test; p values < 0.05 were considered statistically significant.

RESULTS

Distribution of FGF-2 Intratracheal Administration in the Rat

Figure 1 shows a summary of the FGF-2 remaining after intratracheal administration in the rats. Radioactivity remaining after intratracheal administration decreased with time. In the FGF solution group, radioactivity count remaining at 24 h, 72 h, and 7 days was 35.4 [+ or -] 3.6, 5.4 [+ or -] 2.5, and 1.2 [+ or -] 0.17, respectively. In the FGF microsphere group, remaining levels of FGF-2 were significantly higher than in the FGF solution group (p = 0.003). Radio-activity remaining at 24 h, 72 h, and 7 days was 50.2 [+ or -] 10.5, 13.7 [+ or -] 1.6, and 2.9 [+ or -] 1.63, respectively. These results suggest that the controlled-release microsphere system increased the effectiveness of FGF-2.

[FIGURE 1 OMITTED]

Pulmonary Function Tests in the Canine Experiment

Body weights did not differ between the FGF - group and the FGF + group: 11.4-1.3 kg vs 11.8 [+ or -] 1.9 kg, respectively. There were no differences in body weight 4 weeks after elastase administration and 4 weeks after treatment (FGF - group, 11.6 [+ or -] 1.2 kg vs 11.5 [+ or -] 1.5 kg; FGF + group, 11.5 [+ or -] 2.1 kg vs 11.0 [+ or -] 2.3 kg). There was no evidence of serious side effects, including thrombocytopenia, anemia, or renal dysfunction, after FGF-2 administration.

Data for Pa[O.sub.2] are shown in Figure 2, top, A. There were no differences in any parameters between the two groups at baseline and 4 weeks after elastase administration: FGF-, 95.5 [+ or -] 7.3 mm Hg vs 85.5 [+ or -] 3.0 mm Hg; FGF +, 92.8 [+ or -] 4.2 mm Hg vs 88.7 [+ or -] 3.4 mm Hg, respectively. Elastase-induced emphysema models had significantly lower Pa[O.sub.2] values at 4 weeks after elastase administration than at the baseline (p = 0.015). Four weeks after treatment, Pa[O.sub.2] in the FGF + group was significantly higher than in the FGF - group: 83.7 [+ or -] 4.6 mm Hg vs 95.3 [+ or -] 5.9 mm Hg (p = 0.036).

[FIGURE 2 OMITTED]

The intratracheal cavity was inflated at different pressures (5 to 70 cm [H.sub.2]O). In the elastase-induced emphysema models, the P/V curve was shifted upward compared with the baseline. The curve in the FGF - group continued to shift upward, whereas that in the FGF + group shifted downward (Fig 2, bottom, B). The FGF + group appeared to exhibit better recovery than the FGF - group in terms of expiratory capacity with intratracheal pressure of 40 cm [H.sub.2]O (0.88 [+ or -] 0.20 L vs 0.79 [+ or -] 0.18 L), although the difference was not statistically significant (p = 0.72). These results suggested pulmonary functional recovery in the FGF + group.

Dynamic Contrast-Enhanced MRI

MRI scans of pulmonary perfusion allowed the bolus of contrast agent to be followed through the superior vena cava, right atrium and ventricle, pulmonary arteries, lung parenchyma, pulmonary veins, left heart, and systemic arteries. At 5 s, the pulmonary arterial tree could be visualized beyond the segmental branches. A diffuse flash on the lung parenchyma was then observed, followed by a gradual increase in signal intensity over the next 20 s (Fig 3). In the elastase-induced emphysema models, the signal intensity was visually lower in the left lung. The left signal intensity was improved in the FGF + group. The difference between the left and right signals was maximal at 20 s. The signal intensity curves during the 60-s scan time are demonstrated in Figure 4. At the baseline, the levels of the signal intensity curves were the same in the right and left lungs. The signal intensity curve in the elastase-induced emphysema models declined in the left lung compared with the right (Fig 4, top right, B), and the signal intensity curve showed a sharply marked first-pass effect of enhancement with a significant lower-intensity peak on the pathologic side. The signal intensity curve in the FGF + group improved significantly in the left lung (Fig 4, bottom right, C). The flow-volume ratios at baseline were 0.99 [+ or -] 0.09. and 1.00 [+ or -] 0.04 in the FGF- group and the FGF + group, respectively (Fig 5). Four weeks after elastase administration, the flow-volume ratio was significantly lower than baseline: FGF - group, 0.70 [+ or -] 0.06; FGF + group, 0.69 [+ or -] 0.07. Four weeks after treatment, dynamic MI/I revealed significant improvement in the FGF + group. The flow-volume ratio in the FGF + group was significantly improved after FGF-2 treatment, while the FGF - group score was the same as, or worse than, that in the clogs assessed before treatment: FGF -, 0.69 [+ or -] 0.05; FGF +, 0.88 [+ or -] 0.06 (p = 0.0041).

[FIGURES 3-5 OMITTED]

Lung Volumes and Histologic Findings

A summary of the effects of FGF-2 on lung volume throughout the study period is presented in Figure 6. In the animals of the FGF - group, the left lung was overinflated compared with the control group. There were no changes in right lung volumes among the three groups. The left lung volume in the FGF + group was reduced compared to the FGF - group, although the difference was not statistically significant: control, 380.0 [+ or -] 82.9 [cm.sup.3]; FGF -, 442.9 [+ or -] 50.4 [cm.sup.3]; FGF +, 402.8 [+ or -] 71.8 [cm.sup.3]. At autopsy, the tissue destruction caused by the elastase was associated with physiologic changes and a marked reduction in the surface area available for gas exchange (Fig 7). In the FGF + group, the mean size of alveoli was closer to control group than that of the FGF - group. There were no significant differences in right lung Lm levels among the three groups. Left lungs that had received elastase had a significantly increased Lm compared with the control group: control, 52.3 [+ or -] 2.8 [micro]m; FGF -, 71.6 [+ or -] 4.0 [micro]m; FGF +, 63.5 [+ or -] 3.2 [micro]m. Lm levels in the control group and the FGF - group were significantly different (p < 0.001). The Lm value for the FGF + group appeared to indicate better recovery than in the FGF - group (p = 0.02).

[FIGURES 6-7 OMITTED]

DISCUSSION

Growth factors and biological regulators have been evaluated experimentally with respect to their potential usefulness in promoting pulmonary parenchymal regeneration in emphysema. (9-11) Reports (11,14) have suggested that growth factors play an important role in fetal lung development in both rodents and humans. FGF is a member of the heparin-binding polypeptide family. It is widely distributed and has been identified in many tissues of neuroectodermal and mesodermal origin. FGF acts as an angiogenic molecule in vitro, while in vivo it stimulates smooth muscle cell growth, wound healing, and tissue repair. (12) FGF-2 is one of the most powerful angiogenic factors known and is indispensable for lung development and branching morphogenesis. Studies (13,14) of the expression of FGF-2 and receptors in the developing fetal rat lung have shown that FGF-2 immunoreactivity is localized to ceils of the airway epithelium, basement membranes, and extracellular matrix. However, although these characteristics of FGF-2 indicate that it would be a potent promoter of pulmonary functional recovery in emphysema, its biological half-life is reported to be < 50 min, which is too short a time to maintain a sustained response. In addition, endothelial cells take almost 1 day to begin to respond to FGF-2 stimulation. (15,16) Hence, the simple administration of free FGF-2 results in few of the desired biological activities such as angiogenesis, branching morphogenesis, and pulmonary functional regeneration. In fact, some researchers (17) have reported that FGF-2 administration produces insufficient angiogenesis to induce revascularization and subsequent airway healing. We used controlled-release microspheres as vehicles for more effective induction of angiogenesis by FGF-2.

When preparing microspheres, FGF-2 is incorporated into the hydrogel mainly as a result of physicochemical and electrical interactions between FGF-2 and the acidic gelatin, similar to the processes observed during hydrogen bonding and hydrophobic interactions. (4) Once incorporated, it is likely that the FGF-2 will be released from the gelatin hydrogel only when the hydrogel is enzymatically degraded to water-soluble gelatin fragments in vivo. A potential problem with this delivery system is therefore that FGF-2 release can only be controlled by changing the in vivo degradability of the gelatin hydrogels. This can be achieved by manipulating the water content of the hydrogels during their preparation. (4,5) We considered whether the microspheres could function as a drug delivery system in the airway and whether they had any unexpected side effects in the lung. In this in vivo study in rats, we compared the time profiles of the radioactivity remaining after intratracheal administration of [sup.125]I-labeled--FGF-2 either incorporated with an acidic gelatin hydrogel or as an aqueous solution. The gelatin hydrogel yielded a higher radioactivity count than the aqueous solution, thus confirming the effectiveness of the gel as a controlled-release mechanism. The controlled-release microspheres expanded the effects of FGF-2 and prolonged its biological half-life in vivo. There was no evidence of serious complications including infection, atelectasis, allergy, and hemorrhage after gelatin hydrogels microspheres administration in this study. However, IV injection of hydrogels has a risk of vascular infarction. Therefore, gelatin microspheres cannot administer by the IV route.

Many groups have researched the lung parenchymal regeneration and alveolar septation for the chronic obstructive pulmonary diseases. (9,11,18) The mechanisms underlying regeneration and alveolar septation in the respiratory organs are still unclear, and it was not possible to determine whether FGF-2 treatment induced an increase in the number of alveoli or in alveolar septation. To date, we have found that FGF-2 treatment for COPD improves pulmonary function in the beagle dogs. The improvement in arterial oxygen gas data can be explained by the following: FGF-2 treatment led to a volume reduction in the affected lung and improvement of blood flow; subsequently, alveolar gas pressure also improved. Because oxygen uptake into the blood is dependent on the presence of a difference between the alveolar and capillary oxygen pressures, these improvements led to an improvement in the ventilation/perfusion shunt and in the gas exchange ability of the lung. In fact, little hypoxemia was observed after FGF-2 treatment, and the respiratory performance status of all dogs was improved, with no evidence of respiratory insufficiency.

There are some difficulties in attempting to achieve parenchymal regeneration and alveolar septation by the introduction of FGF-2 alone, because in the extracellular environment many growth factors and biological regulators interact with each other to produce parenchymal regeneration or alveolar septation. (13,19) It is difficult for tissue regeneration to occur in regions where blood flow is poor: pulmonary blood flow recovery is indispensable for lung regeneration and wound healing. Our present results indicate that the powerful angiogenic effect of FGF-2 induced both pulmonary revascularization and pulmonary vasodilation in the canine emphysema models, and suggest that this treatment may improve the symptoms of emphysema in humans. The magnitude of the physiologic improvement seen in response to FGF-2 treatment in this experimental model would be expected to benefit patients with severe emphysema to combine the intratracheal FGF-2 treatment and LVRS. We believe that intratracheal FGF-2 treatment has effective potential for emphysema, and that it will become one of the standard therapies for severe emphysema patients.

In conclusion, intratracheal administration of FGF-2 induced an increase in pulmonary blood flow in the damaged lung and volume reduction in the emphysematous lung. These changes led to an improvement in the ventilation/perfusion shunt and thus introduced the pulmonary functional recovery. The use of a controlled-release microsphere delivery system increased the effectiveness of FGF-2.

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* From the Institute for Frontier Medical Sciences (Drs. Nakamura, Toba, Takahashi, Kushibiki, Tabata, and Shimizu), Kyoto University, Kyoto; and Division of Surgical Oncology (Dr. Morino), Nagasaki University School of Medicine, Nagasaki, Japan.

FGF-2 was provided by Kaken Pharmaceutical Company, Tokyo, Japan; and gadopentetate dimeglumine was provided by Magnevist, Nihon Schering, Osaka, Japan.

Manuscript received September 2 2004. revision accepted January 3, 2005.

Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (www.chestjournal. org/misc/reprints.shtml).

Correspondence to: Shigeyuki Morino, MD, Department of Bioartificial Organs, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawaharacho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan; e-mail: morinos@frontier.kyoto-u.ac.jp

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