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Original Investigations

Fibroblast Growth Factor-1 Therapy for Advanced Emphysema—A New Tissue Engineering Approach for Achieving Lung Volume Reduction

Ingenito, Edward P. MD, PhD*; Tsai, Larry W. MD*; Suki, Elizabeth MD, PhD; Hoffman, Andrew DVM, DVSc

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Abstract

Several nonsurgical bronchoscopic approaches to achieve lung volume reduction for the treatment of advanced emphysema have recently been developed and are currently undergoing clinical trials.1–3 If successful, these treatments would make volume reduction therapy available to emphysema patients suffering from medically refractory disease with substantially less morbidity and mortality than conventional lung volume reduction surgery.4,5 Our laboratory has developed a biologic method for producing nonsurgical lung volume reduction using injectable, biodegradable reagents to remodel and shrink damaged lung.6 This approach, refined in animal models, has completed phase 1 clinical testing in patients, and seems to be both safe and potentially effective in selected individuals with heterogeneous upper lobe disease.7

Basic science investigations characterizing the cellular and molecular processes underlying biologic lung volume reduction (BLVR) have provided a clearer understanding for its mechanistic basis.8 These investigations have shown that BLVR is associated with a characteristic pattern of endogenous growth factor release at sites of treatment. Sequential instillation of BLVR reagents containing trypsin, thrombin, and fibrinogen into the lung caused local release of fibroblast growth factor-1 (FGF-1), transforming growth factor-β1 (TGF-β1), and platelet derived growth factor (PDGF). These data have provided a “biologic blueprint” for designing therapeutic approaches to remodel lung tissue using specific growth factors.

We hypothesized that supplementing the pro-fibrotic milieu established by TGF-β and PDGF with additional mitogenic stimulation would potentiate local mesenchymal cell expansion, resulting in more effective tissue contraction and lung volume reduction. To investigate this hypothesis, a fibrin hydrogel containing the mitogenic growth factor FGF-1 complexed to a glycosaminoglycan carrier molecule was injected into the lungs of sheep with experimental heterogeneous emphysema.9,10 Animals were followed for 1 month, and clinical, radiographic, physiologic, and pathologic outcome parameters were assessed. This pilot study indicates that hydrogels containing FGF-1 can be administered bronchoscopically to produce site-specific tissue remodeling and contraction of the emphysema lung with no short-term adverse effects.

METHODS

Protocol

Studies were performed under a protocol approved by the Tufts Cummings School of Veterinary Medicine Institutional Animal Care and Utilization Committee. An experimental model of heterogeneous emphysema was developed as follows. Six healthy female sheep (weight 56±13 kg) received aqueous buffered elastase (600 IU/L of 0.1 M Tris-HCl, pH 8.0; Worthington Chemical) bi-weekly for 6 weeks through a bronchoscope (20 mL per subsegment) into selected caudal lung lobes. Treatments were administered in awake, sedated animals (IV midazolam, 0.5 mg/kg) receiving supplemental oxygen by facemask while monitoring vital signs.

Four weeks after completion of elastase instillation, animals underwent computed tomography (CT) scanning to document heterogeneous emphysema, and identify potential BLVR target sites. Two animals were assigned to control therapy, and received hydrogel without growth factor. The other 4 animals received experimental therapy with hydrogel containing growth factor.

Treatments were administered under general anesthesia (IV bolus propofol 1 mg/kg, midazolam 0.5 mg/kg, and ketamine 1.5 mg/kg followed by maintenance infusion of IV propofol 0.5 to 1 mg/min) with animals orotracheally intubated and mechanically ventilated. Pretreatment baseline CT images were obtained on each animal at a transpulmonary distending pressure of +25 cm H2O [defined as total lung capacity (TLC)]. Animals then received either control or experimental treatment at 4 sites in each lung, and were allowed to recover.

Activity level, vital signs, and peripheral oxygen saturation levels were documented daily for the first 3 days posttreatment, and then weekly thereafter for 4 weeks. Body weight was recorded at baseline and at 4 weeks prior to sacrifice.

Response to treatment was assessed at the 4-week time point radiographically and pathologically. Necropsy findings were documented photographically, and tissues were harvested and fixed in 10% neutral buffered formalin. Samples from treatment sites were processed for histopathology and stained with hematoxylin and eosin, and Mason trichrome. Selected specimens were analyzed by immunohistochemistry.

FGF-1 Hydrogel Formulation

Experimental therapy (FGF-1 containing hydrogel) involved treatment of subsegmental target sites with 3 reagents administered sequentially. The bronchoscope was placed in a wedge position in the orifice of the subsegmental airway selected for treatment. Ten milliliters of Enzymatic Primer (0.25% trypsin containing 0.03% ethylenediaminetetraacetic acid in buffered saline) was injected and left in place for 2 minutes followed by 1 minute of suction. Ten milliliters of washout solution containing trypsin neutralizer in RPMI-1640 media was then injected and left in place for 30 seconds, followed by 30 seconds of suction. A dual lumen catheter was passed through the bronchoscope and the tip was positioned 1 to 2 cm beyond the end of the scope. Ten milliliters of fibrinogen solution (26.6 mg/mL human fibrinogen) containing FGF-1 (750 ng/mL) complexed to chondroitin sulfate (5 mg/mL) with tetracycline (5 mg/mL) dissolved in RPMI-1640 media was injected simultaneously with 1 mL of thrombin crosslinker (4 U thrombin/mg of fibrinogen in 40 mM CaCl2) to generate a polymerized hydrogel in situ. The bronchoscope was left in place for 30 seconds after hydrogel instillation to ensure polymerization, and then repositioned at the next treatment site.

Control therapy (n=2 animals) was administered similar to experimental therapy except that control hydrogels did not contain any FGF-1. Primer, washout, and hydrogel were otherwise the same.

CT Assessment

CT scans were obtained with a Picker 5000 CT scanner using 5-mm collimation and 2.5-mm overlapping reconstruction algorithm. All scans (baseline and posttreatment) were obtained at transpulmonary distending pressures of +25 cm H2O. Lung volumes were determined by digital integration from lung apex to the most caudal portion of the diaphragm using Emphy LXJ software (Licensed from University of British Columbia). CT images were also evaluated qualitatively for evidence of pneumonia, pleural changes, lung abscess formation, localized scarring, and mediastinal soft tissue abnormalities.

Necropsy, Histopathology, and Immunohistochemistry

A thorough inspection of the thoracic contents including the lung, mediastinum, and visceral and parietal pleural surfaces was performed at necropsy. Noteworthy findings were documented photographically. Tissue specimens of treatment and control sites from the lung were obtained, as well as samples from liver, kidney, heart and spleen. Samples were fixed in 10% neutral buffered formalin, embedded in paraffin, cut, and stained with hematoxylin and eosin, and Mason trichrome using standard histologic protocols. Additional cut sections were evaluated by immunostaining for epithelial and fibroblast cell markers, and extracellular matrix components. Either fluorescent or enzymatic (peroxidase) detection systems were used to optimize sensitivity and specificity.

RESULTS

Clinical Responses

Bronchoscopic lung volume reduction using an FGF-1–based hydrogel system was well tolerated in sheep with heterogeneous experimental emphysema. During administration, the hydrogel polymerized rapidly and spillage back into the central airways was not observed. Oxygen saturation levels remained greater than 90% in all animals throughout the course of treatment on mechanical ventilator settings of assist-control mode ventilation, rate of 12 breaths/min, tidal volume of 10 mL/kg, FiO2=0.6, and PEEP=0 cm H2O. All animals resumed room air ventilation within 15 minutes of discontinuation of anesthesia.

Elevated body temperature (defined as T>103.5°F) was observed in 2 experimental therapy animals and 1 control therapy animal during the first week after treatment. In all cases, fever was observed during the first 2 days posttreatment, and resolved spontaneously without intervention. Activity levels, body weights, respiratory rates, and heart rate were unchanged from pretreatment baseline in all animals throughout follow-up.

CT Image Results

Elastase treatment was associated with development of mild-to-moderate emphysema in the caudal lung lobes bilaterally. Several animals demonstrated focal tissue destruction that was visible by CT imaging. Examples of such lesions are shown in Figure 1 (left panels).

FIGURE 1.
FIGURE 1.:
Pretreatment CT scans (left) and 1-month posttreatment CT scans (right) showing evidence of atelectasis and volume loss in response to BLVR using FGF-1. Treatment areas are as outlined.

Treatment with hydrogel containing FGF-1/chondroitin sulfate complex was associated with remodeling, scar formation, and volume loss localized to sites of treatment (Fig. 1, right panels). Lung volumes measured at TLC (+25 cm H2O transpulmonary inflation pressures) were reduced in all experimental therapy animals at 1 month (Fig. 2). Volume reduction [(baseline volume−lung volume at 4 weeks)/baseline volume] after treatment at 8 subsegmental caudal sites was 12±5% (range 5.0% to 16.3%), equivalent to an average reduction of 1.5% of TLC (52 mL) per treatment site. Two bullous lesions that had developed after elastase treatment showed complete collapse and involution 1 month after BLVR treatment with FGF-1/chondroitin hydrogel.

FIGURE 2.
FIGURE 2.:
Lung volumes measured by digital integration of CT images at baseline (pretreatment) and at 1-month posttreatment. Reductions were observed in all animals, and ranged from 5% to 16%.

Control therapy was associated with substantially smaller reductions in lung volume than experimental therapy at follow-up. Lung volumes increased slightly in one animal (+3%) and decreased slightly in another (−5%).

Necropsy Findings

Important necropsy findings were limited to the chest cavity. Two of 4 experimental therapy animals had small dorsal pleural adhesions that were not associated with pleural fluid collections. There was no abnormal mediastinal adenopathy (defined as nodes >1 cm in diameter), subpleural hydrogel collections, or parenchymal abscess formation. BLVR with FGF-1 complexed to chondroitin sulfate produced scarring with visceral pleural contraction at sites of treatment. Representative examples of necropsy findings from 2 animals (nos. 140 and 13) are shown in Figure 3. On cut section, 2 (out of 32) treatment sites demonstrated small amounts of residual hydrogel that were associated with surrounding scar tissue.

FIGURE 3.
FIGURE 3.:
Necropsy photos of sheep nos. 140 and 13 show experimental treatment sites 4 weeks after instillation of fibrin hydrogels containing FGF-1 complexed to chondroitin sulfate. Scar formation and tissue contraction is readily apparent. Necropsy photos of sheep no. 17 show Control treatment sites that had received similar treatment but without FGF-1. Minimal scarring is observed at Control sites relative to Experimental sites at 4-week follow-up.

Control therapy was associated with minimal scar formation at the site of treatment. Necropsy findings from a control animal (no. 17) are shown in Figure 3.

Histopathology and Immunohistochemistry

Potential cellular sites for FGF-1 response were identified in healthy and emphysema sheep lung by performing immunostaining using an antibody with reactivity against fibroblast growth factor receptor (FGFR) 1 and 2. These 2 receptors, present on ciliated epithelial cells, fibroblasts, alveolar type II cells, and pulmonary endothelial cells, were readily detectable in healthy sheep tissue, and in untreated sites of lung from animals with experimental emphysema.11 Receptor distribution identified by immunoperoxidase staining is shown in Figure 4.

FIGURE 4.
FIGURE 4.:
Immunoperoxidase staining of control lung tissues for FGFR 1/2. Numerous cells in the lung interstitium (top panel) stain positive for the FGFR 1/2. Alveolar type II cells and interstitial fibroblasts are highlighted. Ciliated bronchiolar epithelial cells (bottom panel) also stain positive for the receptor.

Histologic evaluation of treatment sites demonstrated extensive tissue remodeling with formation of organized scar 4 weeks after dosing with FGF-1/chondroitin sulfate hydrogel. Hematoxlyin and eosin staining showed loss of normal parenchymal architecture and replacement by extracellular matrix (Fig. 5). Trichrome staining confirmed that the remodeled tissues primarily contained collagen (Fig. 5).

FIGURE 5.
FIGURE 5.:
Hematoxylin and eosin-stained lung tissue from a FGF-1 BLVR treatment site 4 weeks after dosing (upper panel). Normal lung architecture seen at bottom right of top panel has been largely replaced by acellular scar tissue. Tissues stained with Mason trichrome (lower panel) show blue-staining fibers, consistent with newly organizing collagen.

Additional staining for cell markers was performed to identify cell types participating in the remodeling response. Staining with fluorescent antibodies to epidermal growth factor receptor was performed to assess epithelial cell proliferation. Control stains were performed on tissues taken from sites of untreated lung in the same animal. Examples are shown in Figure 6. Epidermal growth factor receptor staining is readily visible along the alveolar septa of control tissue from an untreated site, although minimal staining is present in areas of remodeled, contracted scar tissue, despite substantial cellularity in these regions as demonstrated by nuclear (blue DAPI) staining.

FIGURE 6.
FIGURE 6.:
Fluorescent staining for epithelial cell growth factor receptor (bright green) in untreated tissue (top panel) and tissue from a site of treatment (bottom panel) in the same animal. Epithelial cells are apparent along the alveolar septa in control tissue. Tissue from treatment sites shows few epithelial cells, which are localized to the lower left hand portion of the bottom panel. Non-epithelial cells, identified by nonspecific nuclear DAPI staining (blue), are present throughout the remodeled tissue. Autofluorescence associated with collagen is also present.

Immunoperoxidase staining for vimentin was performed to facilitate identification of mesenchymal cells (Fig. 7). Samples from animals receiving control treatment showed a typical pattern of staining, whereas samples from treatment sites receiving FGF-1/chondroitin complex demonstrated extensive staining, indicating a transition from epithelial cell-dominant to mesenchymal cell-dominant tissue. To further characterize the phenotype of cells at treatment sites, α-smooth muscle actin (ASMA) staining was performed (Fig. 8). The pattern of staining was similar to that observed for vimentin, with limited ASMA staining in control parenchyma, but extensive ASMA staining in treated tissues in association with remodeling and parenchymal collapse, confirming the presence of myofibroblasts. Double staining for the cell proliferation antigen ki67 showed that a subfraction of ASMA-positive cells were actively proliferating at remodeled treatment sites 1 month after FGF-1 exposure.

FIGURE 7.
FIGURE 7.:
Immunoperoxidase staining (dark brown) for vimentin at site of tissue damage in experimental emphysema lung of animal receiving control endoscopic volume reduction therapy (no FGF-1; top panel), and a site from an animal receiving endoscopic volume reduction therapy containing FGF-1. Staining for vimentin after control therapy shows typical pattern associated with interstitial fibroblasts along alveolar septae. Collapse has largely resolved after 1 month. By contrast, FGF-1 treatment is associated with complete loss of alveolar structure and extensive staining for vimentin throughout remodeled tissue, which is collapsed and contracted.
FIGURE 8.
FIGURE 8.:
Immunoperoxidase staining (dark brown) for ASMA, a marker for smooth muscle and myofibroblasts, in control treated tissue (no FGF-1, top left panel) and tissue from a site that received treatment with FGF-1 (top right panel). A typical staining pattern for ASMA is seen in control tissues. By contrast, extensive staining for ASMA is seen throughout remodeled tissue at sites of treatment exposed to FGF-1 a month earlier. The diffuse staining throughout the tissues is consistent with the presence of myofibroblasts at the site of treatment.

DISCUSSION

Previous studies have shown that polycations complexed to glycosaminoglycans can be used to safely and effectively remodel emphysema lung to achieve site-directed tissue contraction, producing therapeutic lung volume reduction.6 An analysis of the biology of this response subsequently defined a pattern of growth factor release that seems to modulate this remodeling process. That analysis indicated that several endogenous profibrotic growth factors, including TGF-β and PDGF, were increased during the initial stages of the BLVR response. Based upon their known effects, it was felt that they likely contributed to the development and maturation of scarring responsible for therapeutic volume loss. The present study was designed to test whether the addition of the mitogen, FGF-1, a growth factor capable of stimulating fibroblast proliferation in vitro, could be used to facilitate the tissue remodeling required to achieve effective BLVR.

Testing was performed in an experimental large animal model of emphysema in which tissue destruction was generated through bi-weekly intrabronchial instillation of elastase over 6 weeks. This model is similar to the one previously characterized by our group,3 except that papain was replaced by elastase in an attempt to reduce local inflammation and scarring. The model used in this study was associated with substantially less fibrosis than the papain model, and provided radiographically identifiable target areas of tissue destruction (Fig. 1) for evaluating treatment effectiveness.

Administration of hydrogel containing FGF-1/chondroitin sulfate to target sites within the lung was well tolerated, with no obvious systemic side effects. Two of 4 experimental therapy animals, and 1 of 2 control therapy animals experienced transient fever after endobronchial treatment. Vital signs, activity levels, and body weights were otherwise stable throughout follow-up, and all animals seemed healthy at the time of sacrifice. None of the animals demonstrated clinical signs of respiratory distress during follow up.

CT scanning performed 4 weeks after treatment confirmed the development of dense organizing scar at sites of hydrogel instillation. Tissue remodeling, as indicated by the development of localized areas of collapse, increased tissue density, and/or formation of linear scarring was apparent at all 8 treatment sites in each experimental therapy animal. Compared with prior BLVR formulations that have used polycations to achieve endoscopic volume reduction through tissue engineering principles, the present FGF-1 formulation tested here produced more consistent responses.2,3,6 Tissue collapse and remodeling remained localized to sites of treatment within the lung. As assessed by CT imaging, scar formation was not apparent either globally throughout the lung, or at sites extending beyond those targeted for therapy. The remodeling process was associated with consistent reductions in lung volume measured at iso-inflation pressure (+25 cm H2O distending transpulmonary pressure). Volume rendering performed by digital integration of CT images from lung apex to the caudal edge of the diaphragm demonstrated reductions in TLC of greater than 10% in 3 of 4 animals with an average reduction of 12%.

Necropsy findings confirmed that hydrogels containing FGF-1 produced organized, contracted scarring responses at sites of treatment within 4 weeks. Tissue contraction and volume loss at treatment sites was apparent by visual inspection of the visceral pleural surface of necropsy specimens, as illustrated in Figure 3. Histopathologic examination of treatment sites confirmed that normal parenchyma had been replaced by scar tissue containing collagen and fibroblasts. It is interesting that treatment sites were not associated with epithelial cell proliferation, because bronchiolar and alveolar epithelial cells stained positive for FGF receptors (Fig. 4), and FGF-1 is pleotropic, capable of binding to all FGF receptor isoforms.12,13 The basis for the apparent bio-selectivity of FGF-1 in this setting was not characterized in the present study, but could relate to any of several factors. Pretreatment of target sites with trypsin could bias the subsequent biologic response in favor of fibroblast proliferation by cleaving FGF receptors on surface epithelial cell surfaces while causing partial epithelial cell detachment, exposing interstitial fibroblasts below. Tryspin also has been shown to activate local PAR receptors, leading to release and de novo synthesis of TGF-β.14–16 Increased levels of TGF-β, which can cause epithelial cell apoptosis, and epithelial-mesenchymal transformation, could bias the local mitogenic effects of FGF-1 toward fibroblast-specific proliferation.17 The hydrogel, which contains both fibrin and fibronectin, possesses RGD binding sites capable of attracting fibroblasts and circulating fibrocytes to areas of treatment.18–20 Finally, it is possible that prior treatment of these sites with elastase during model development may have contributed to the specificity of the FGF-1 response for fibroblasts by promoting preexisting tissue damage.

Although the hydrogel formulation tested here contains multiple constituents including fibrin and fibrin split products, thrombin, fibronectin, RPMI 1640 media, and tetracycline, control animals treated with the same formulation supplemented with chondroitin sulfate but not FGF-1 demonstrated substantially attenuated responses. Lung volumes in these animals after 4 weeks were not reduced compared with baseline measurements, and minimal scarring and pleural contraction were observed at necropsy (Fig. 3). Furthermore, histologic evaluation of control samples showed substantially less tissue remodeling and extracellular matrix deposition than experimental therapy animals, and qualitatively fewer fibroblasts at sites of treatment. These findings indicate that FGF-1 is essential for facilitating the rapid scarring, tissue contraction, and physiologic volume reduction observed here, and that the bio-modulatory effects of fibrin and fibronectin, although capable of promoting the very mild scar formation observed in control animals, are ineffective for achieving physiologically relevant lung volume reduction.

FGF-1, acidic fibroblast growth factor, has generally not been used as a therapeutic biologic because of pharmacokinetic and pharmacodynamic considerations. FGF-1 is nonselective in its binding to FGF receptors, and has an extremely short half-life in vivo.21,22 For these reasons, FGF-2 has generally been used in the development of biopharmaceuticals in which FGFR activation is intended for therapeutic applications such as endothelial cell proliferation.23 Prior studies have demonstrated that it is possible to stabilize FGF-1 in vivo by precomplexation with glycosaminoglycans. In most instances, heparin sulfate has been used as the stabilizing substance as it substantially alters FGF-1 degradation profiles, and seems to markedly increase its bioactivity by facilitating binding to the FGF receptor.12,24 In the present study, chondroitin sulfate was selected as the stabilizing agent of choice because of concerns regarding the potential anticoagulant effects of heparin sulfate absorbed into the systemic circulation posttreatment. In vitro studies performed using primary sheep lung fibroblasts confirmed that FGF-1 complexed to chondroitin sulfate was capable of promoting cell proliferation. The formulation tested here using relatively large amounts of growth factor (750 ng/mL) provided consistent and reproducible biologic and physiologic responses in the lung. FGF-1 complexed to heparin sulfate was not tested in this study, nor was FGF-2. It is possible that at appropriately selected doses, FGF-1 or FGF-2 could be used with either chondroitin sulfate or heparin sulfate with equivalent safety and effectiveness in the lung.

Although results presented here suggest that FGF-based hydrogel therapies for producing lung volume reduction may hold promise, several aspects of this response require further evaluation before considering clinical application of this approach. Although the majority of treatment sites at 1 month presented as linear organizing scars, 2 responses (out of 32) demonstrated more extensive parenchymal scarring and collapse with fibrosis throughout large areas of parenchymal. One such area is depicted in the top right panel of Figure 1. Necropsy evaluation of this region confirmed the presence of firm scar tissue. Histopathology demonstrated fibroblasts that were ki67 positive replacing normal lung tissue, indicating ongoing proliferation. Although it is unlikely that this represents an ongoing mitogenic effect of residual FGF-1 remaining from the initial treatment, it is possible that paracrine and/or autocrine effects related to this initial treatment are responsible for perpetuating this response. Additional studies are required to further address this question of local fibroblast proliferation. Furthermore, although no hepatic, renal, cardiac, or splenic toxicity was observed during histologic inspection of tissue from these organs at 1 month, assessment of systemic toxicity at later time points is also required.

Despite these limitations, initial results presented here suggest that FGF-1 complexed to a carrier glycosaminoglycan can effectively and safely promote collapse of damaged areas of lung as a treatment for emphysema. BLVR therapy using FGF-1 was well tolerated during 1-month follow-up, with no clinical or pathologic evidence of systemic toxicity. Animals receiving experimental therapy demonstrated scarring and tissue contraction localized to sites of treatment, whereas control animals receiving hydrogel without FGF-1 had minimal tissue responses, and no measurable reductions in lung volume. Longer term follow-up and additional dosing studies are required to further characterize the lung's response to FGF-1 treatment, and determine the correct dosing regimen to optimize therapeutic response and in vivo safety. Nevertheless, these studies indicate that a new generation of tissue-engineering reagents containing growth factors complexed to carrier molecules may soon become available for promoting lung volume reduction endoscopically.

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Keywords:

emphysema; tissue engineering; lung volume reduction; fibroblast growth factors; FGF-1

© 2006 by Lippincott Williams & Wilkins, Inc.