Abstract
The role of platelet-derived growth factor (PDGF) in the development of obliterative bronchiolitis (OB) as a manifestation of chronic rejection was investigated in the heterotopic rat tracheal allograft model. An increase in intragraft PDGF-R α and -R β mRNA expression, and in PDGF-AA and -R α immunoreactivity, was demonstrated during the progressive loss of respiratory epithelium and airway occlusion in nontreated allografts compared with syngeneic grafts. Treatment with CGP 53716, a protein-tyrosine kinase inhibitor selective for PDGF receptor, alone and in combination with suboptimal doses of cyclosporin A, significantly reduced myofibroproliferation and the degree of OB by more than 50%. CGP 53716 did not affect airway wall inflammatory cell proliferation, the number of graft-infiltrating CD4+ or CD8+ T cells, ED3+ macrophages, or the level of immune activation determined as IL-2R and MHC class II expression. This study suggests a regulatory role for PDGF, especially for PDGF-AA and -R α , in the development of obliterative bronchiolitis in this model, and demonstrates that inhibition of PDGF receptor protein-tyrosine kinase activation prevents these obliterative changes. Thus, receptor protein-tyrosine kinase inhibitors may provide a novel therapeutic strategy for the prevention of chronic rejection.
Platelet-derived growth factor (PDGF) is a major mitogen for mesenchymal cells such as smooth muscle cells (SMCs) and fibroblasts (reviewed in Reference 1). PDGF ligands consist of disulfide-linked dimers of two polypeptides, the PDGF-A and PDGF-B chains, and can be expressed as homodimers (PDGF-AA and PDGF-BB) or as a heterodimer (PDGF-AB) (1). The three different isoforms bind with different affinities to two related tyrosine kinase receptors, PDGF-Rα and the PDGF-Rβ (2, 3). PDGF-Rβ can bind only the PDGF-B chain, whereas PDGF-Rα binds both A and B chains (3). Binding of PDGF to the extracellular part of either receptor type leads to dimerization of receptor molecules, followed by activation of the receptor protein-tyrosine kinase, and generation of phosphorylation-mediated signals that initiate the biological response (2). Smooth muscle cells (4) and fibroblasts (5) synthesize only the PDGF-A chain, whereas endothelial cells (6, 7) and macrophages (8) synthesize both chains. The PDGF receptors have been localized to SMCs (9), fibroblasts (10), endothelial cells (11), epithelial cells, including respiratory epithelium (12, 13), and macrophages (14).
PDGF may have a pivotal role in fibroproliferative disorders of lungs, including idiopathic pulmonary fibrosis (15), adult respiratory distress syndrome (ARDS) (16), and bronchiolitis obliterans-organizing pneumonia (17). PDGF is upregulated during the development of obliterative bronchiolitis (OB), a manifestation of chronic lung allograft rejection (18), as well as during the development of chronic rejection of kidney (19) and heart (20) allografts, suggesting that PDGF may be involved in the development of fibroproliferative lesions consisting of mesenchymal cells in the allograft vascular wall, interstitium, and airways in chronic rejection.
CGP 53716 is a novel inhibitor of protein-tyrosine kinase activity and highly selective for the PDGF receptor tyrosine kinase in vitro and in vivo (21). The compound is selective for the inhibition of PDGF-mediated events such as PDGF-R autophosphorylation, cellular tyrosine phosphorylation, and c-fos mRNA induction in response to PDGF stimulation of intact cells. In contrast, ligand-induced autophosphorylation of epidermal growth factor (EGF) receptor, insulin receptors, and the insulin-like growth factor I (IGF-I) receptor, as well as c-fos mRNA expression induced by EGF, basic fibroblast growth factor (bFGF), and phorbol ester are insensitive to inhibition by CGP 53716 (21).
Previous studies in heterotopic tracheal allografts demonstrate that epithelial metaplasia and necrosis, mononuclear inflammatory cell infiltration, and induction of proinflammatory cytokines and growth factors leading to proliferation of α-smooth muscle cell actin-positive cells and gradual occlusion of the airway lumen develop like OB in human (22-24). In syngeneic grafts, slight epithelial injury due to ischemia is seen early after transplantation followed by complete recovery of respiratory epithelium, and no obliterative changes are observed (23, 24).
To establish the regulatory role of PDGF in the development of experimental OB in rat tracheal allografts, we determined the induction of intragraft PDGF ligands and receptors at mRNA and protein levels and tested whether the inhibition of the PDGF signal transduction pathway by a protein-tyrosine kinase inhibitor, CGP 53716, would inhibit the development of OB. The data demonstrate a significant suppression of experimental OB by a PDGF protein-tyrosine kinase inhibitor and thus provide evidence that PDGF has a rate-limiting role in this disease process.
Specific pathogen-free, inbred male DA (AG-B4, RT1a) and WF (AG-B2, RT1u) rats weighing 200–300 g and 2–3 mo of age (Laboratory Animal Center, University of Helsinki, Helsinki, Finland) were used. The animals received humane care in compliance with the Principles of Laboratory Animal Care and the Guide for the Care and Use of Laboratory Animals prepared and formulated by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH Publication No. 86-23, revised 1985). Allogeneic heterotopic tracheal transplantations were performed as described (23, 24) from DA to WF rats; syngeneic controls involved transplantations from DA to DA rats. Transplantations were performed under chloral hydrate anesthesia (240 mg/kg, intraperitoneal) and buprenorphine (Temgesic, 0.25 mg/kg, subcutaneous; Reckitt & Colman, Hull, UK) was used for postoperative pain relief. To investigate the induction and localization of PDGF ligand and receptor expression at mRNA and protein levels in experimental OB, syngeneic grafts and allografts were harvested 3, 10, and 30 d after transplantation. To study the effect of the protein-tyrosine kinase inhibitor specific to PDGF receptor, CGP 53716, on the development of OB, the recipients received either (1) vehicle, or (2) CGP 53716, or (3) vehicle and cyclosporin A (CsA), or (4) CGP 53716 and CsA, and the grafts were removed 10 and 30 d after transplantation. CsA was introduced to the study protocol to reduce the cellular immune response and to prevent the rapid development of OB changes. A suboptimal dosage of CsA, 1 (mg/kg)/d, was employed, as optimal immunosuppression with CsA [2–5 (mg/kg)/d] abolishes obliterative changes (23), and could thus make it impossible to demonstrate effects of CGP 53716.
Cyclosporin A (CsA, Sandimmune; Novartis, Basel, Switzerland) was dissolved in Intralipid (200 mg/ml; KabiVitrum, Stockholm, Sweden) to a final concentration of 1 mg/ml and was administered subcutaneously at a dosage of 1 (mg/kg)/d. Whole blood CsA 24-h trough levels were determined using a radioimmunoassay (Sandimmune kit; Novartis). CGP 53716, a PDGF-R protein-tyrosine kinase inhibitor (Novartis), was dissolved in dimethyl sufoxide (DMSO) to a concentration of 200 mg/ml and diluted thereafter 1:20 with 1% Tween in 0.9% NaCl and sonicated. The stock solution and dilutions were prepared daily before administration. The rats received 50 (mg/kg)/d of CGP 53716 by daily intraperitoneal injection starting 24 h before transplantation. This dose has been shown to be well tolerated and effectively inhibits the activity of tumors derived from v-sis- and c-sis-transformed BALB/c cells in BALB/c nude mice (21). Controls received an equal amount of vehicle prepared and administered like the drug. Rats were weighed once a week to ensure exact dosing of the drugs.
The grafted trachea was excised, embedded in Tissue-Tek (Miles, Elkhart, IN), snap-frozen in liquid nitrogen, and stored at −70° C until use. For histological evaluation, frozen sections were stained with Mayer's hematoxylin and eosin (H&E). Histological changes in the respiratory epithelium were evaluated as a percentage of normal respiratory, abnormal cuboidal, and squamous epithelium, or loss of epithelium. Lumenal occlusion was evaluated, using an ocular grid, as the reduction in lumenal area. All analyses were done by two observers in a blind review, and the scores of these two observers were highly correlated (r2 = 0.90). The degree of airway wall inflammation, the cell types of the myofibroproliferative lesion, and the number of proliferating inflammatory and myofibroproliferative cells were analyzed using immunohistochemistry.
Serial frozen sections (4–6 μm) were air dried on silane-coated slides, fixed in acetone at −20° C for 20 min, and stored at −20° C until use. Before immunostaining, the slides were refixed with chloroform and then air dried. After incubation with appropriate 1.5% nonimmune serum (Vector Laboratories, Burlingame, CA) for monoclonal antibodies or polyclonal antibodies, frozen sections were incubated with mouse monoclonal antibodies at room temperature for 30 to 60 min or with rabbit polyclonal antibodies at 4° C for 12 h. The primary antibodies were diluted in phosphate-buffered saline (PBS) with 1% bovine serum albumin (BSA) and appropriate 3% nonimmune serum. With intervening washes in TRIS-buffered saline, the following compounds were administered: biotinylated horse anti-mouse or goat anti-rabbit rat-absorbed antibodies at room temperature for 30 min, and avidin-biotinylated horseradish complex (Vectastain Elite ABC kit; Vector Laboratories) in PBS at room temperature for 30 min. The reaction was revealed by chromogen 3-amino-9-ethylcarbazole (AEC; Sigma, St. Louis, MO) containing 0.1% hydrogen peroxidase, yielding a brown-red reaction product. The specimens were counterstained with hematoxylin and coverslips were aquamounted (Aquamount; BDH, Poole, UK).
To evaluate the expression of PDGF ligands and receptors in the development of OB in the nontreated syngeneic grafts and allografts, the following primary affinity-purified rabbit polyclonal antibodies were used as described (25): an IgG antibody to human recombinant PDGF-AA, recognizing human and rat PDGF-AA (6.7 μg/ml, ZP-214; Genzyme, Cambridge, MA); an IgG antibody to human recombinant PDGF-BB, recognizing human and rat PDGF-BB (10 μg/ml, ZP-215; Genzyme); an IgG antibody to the carboxy terminus of human PDGF-Rα, recognizing human, mouse, and rat PDGF-Rα (0.5 μg/ml, sc-338; Santa Cruz Biotechnology, Santa Cruz, CA); and an IgG antibody to the carboxy terminus of human PDGF-Rβ, recognizing human, mouse, and rat PDGF-Rβ (0.5 μg/ml, sc-339; Santa Cruz Biotechnology). To evalute the effect of different treatment modalities on inflammatory cell subsets and immune activation, allografts were stained using the following antibodies as described (24): mouse monoclonal antibodies directed to rat activated macrophages (ED3; Serotec, Oxford, UK), CD4+ (W3/25; Sera-Lab, Sussex, UK) and CD8+ (OX8; Sera-Lab) T cells, MHC class II common determinant (OX6; Sera-Lab), and IL-2R (PharMingen, San Diego, CA).
Specificity controls were performed using the same immunoglobulin concentration of species- and isotype-matched antibodies; mouse monoclonal IgG1 antibody (X931; Dako A/S, Glostrup, Denmark) and rabbit polyclonal immunoglobulin fraction (X936; Dako A/S) for monoclonal antibodies and polyclonal antibodies, respectively. Additional controls for the specificity of PDGF-AA (recombinant human PDGF-AA homodimer; Genzyme), PDGF-BB (recombinant human PDGF-BB homodimer; Genzyme), PDGF-Rα (control peptide; Santa Cruz Biotechnology), and PDGF-Rβ (control peptide; Santa Cruz Biotechnology) staining involved the use of a working dilution of the affinity-purified polyclonal antibodies after overnight incubation with a 20-fold molar excess of the corresponding peptide antigen.
The immunohistochemical analysis was done in a blind review by two observers, and the score assigned was determined by consensus. The intensity of staining was scored from 0 to 3 as follows: 0, no visible staining; 1, few cells with faint staining; 2, moderate intensity with multifocal staining; and 3, intense diffuse staining of the cells analyzed.
Relative PDGF ligand and receptor gene transcript levels were measured by reverse transcriptase-polymerase chain reaction (RT-PCR) after normalization against levels of the reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Total RNA was extracted from fresh tracheal tissue using guanidine isothiocyanate preparation as described (26). The total RNA yields were estimated by spectrophotometry and confirmed by 1% agarose–TRIS–borate gel electrophoresis.
To identify the optimal PCR conditions for accurate measurement of each gene, the cycle number during which the amount of PCR product was on the linear part of the S slope (on a logarithmic scale) was determined using tracheal samples from each group and by performing the PCR analysis by linearly increasing the cycle number. The analysis was performed for each gene, and linear amplification was found at 35 cycles for PDGF-A, -B, -Rα, and -Rβ and at 33 cycles for GAPDH.
One microgram of total RNA in 10 μl of RNase-free water was reverse transcribed after addition of 10 μl of RT master mix containing the reverse transcriptase, at 37° C for 90 min. The enzyme was inactivated at 95° C for 5 min. cDNA reaction mixture (0.5 μl) was supplemented with PCR master mix containing the sense primer and antisense primer. The initial denaturation/enzyme activation was carried out at 95° C for 10 min and the samples were then cycled 35 times (94° C for 30 s, 55° C for 30 s, 72° C for 30 s) on a GenAmp PCR System 9700 apparatus (Perkin-Elmer, Norwalk, CT). The amount of GAPDH mRNA in tracheal samples was analyzed by a similar protocol, using GAPDH sense and antisense primers and amplification for 33 cycles (94° C for 1 min, 57° C for 1 min, 72° C for 2 min). The following primers were used: PDGF-A sense (5′-AGA AGT ATT GAG GAA GCC ATT CC-3′) and antisense (5′-TCA CCT GGA CCT CTT TCA ATT T-3′) (GenBank accession no. L06894), PDGF-B sense (5′-CTG AGC TGG ACT TGA ACA TGA C-3′) and antisense (5′-CAC TAC TGT CTC ACA CTT GCA GG-3′) (GenBank accession no. Z14117), PDGF-Rα sense (5′-GAG AAG ATT GTG CCG CTG AGT-3′) and antisense (5′-CAC ACT GAA GGT TCC GTT GAA G-3′) (GenBank accession no. M63837), PDGF-Rβ sense (5′-TCG TCC TCA ACA TTT CGA GC-3′) and antisense (5′-TCA TAG GGT ACA TGT AGG GGG AT-3′) (GenBank accession no. Z14119), and GAPDH sense (5′-GTC TTC ACC ACC ATG GAG AAG GCT-3′) and antisense (5′-TGT AGC CCA GGA TGC CCT TTA GTG-3′) (GenBank accession no. M17701). Gene transcript levels for all samples for a single gene were amplified simultaneously, and each PCR analysis was performed three times. For each experiment, the negative controls, for which water was used instead of cDNA or reverse transcriptase was omitted during cDNA synthesis, were performed. The PCR samples were electrophoresed through 1.5% agarose. The gels were subsequently dried (LKB 2003 slab gel dryer; LKB, Bromma, Sweden), exposed to an imaging plate (Fuji Photo Film, Tokyo, Japan) and then quantified using a Fuji BAS 1500 phosphoimager (Fuji). The mean values of the three determinations were used for final analysis, and the normalized PDGF ligand and receptor mRNA levels were derived by dividing the mean by the mean of GAPDH mRNA for each tissue sample.
All recipients were injected intravenously with 400 μl of a concentrated solution of bromodeoxyuridine (BrdU; solution of 5-bromo-2′-deoxyuridine [3 mg/ml] and 5-fluoro-2′-deoxyuridine [0.3 mg/ml]; Zymed Laboratories, San Francisco, CA) 3 h before sacrifice. Cell proliferation in frozen sections was revealed by an IgG1 mouse monoclonal antibody to BrdU (M744, diluted 1:20; Dako A/S) and the Vectastain Elite ABC kit method as described above. Before staining, the frozen sections were fixed with buffered formalin for 15 min. After a 10-min wash in PBS, the sections were microwave-treated with 500 W in 0.1 M citrate buffer, pH 6, for 5 min to break the double-stranded DNA, followed by a 10-min wash in PBS. Cell proliferation was measured by counting the number of labeled nuclei in tracheal cross-sections.
All data are expressed as means ± SEM. For two-group comparisons of small sample size, the nonparametric Mann–Whitney U test (Statview 512+ program; Brain Power, Calabasas, CA) was used. For multiple-group comparisons of small sample size, nonparametric Kruskal– Wallis (Statview 512+ program) one-way analysis by ranks was used. The rank sums obtained with the Kruskal–Wallis test were then used for the Dunn test at the significance levels of 5 and 1% (Medstat; Astra Group A/S, Copenhagen, Denmark). In groups with normal distribution and standard variance (F test), one-factor analysis of variance (ANOVA) and Fisher PLSD test (Statview 512+ program) were used at significance levels of 5, 1, and 0.1%. p < 0.05 was regarded as significant.
In syngeneic grafts, the respiratory epithelium was slightly damaged at 3 d, probably owing to ischemic injury before revascularization of the graft. By 10 d, the airway lumen was covered with normal respiratory epithelium. In syngeneic grafts, no myofibroproliferation occurred and the overall histology resembled that of normal nontransplanted tracheas (Figure 1). In nontreated allografts, there was progressive loss of epithelium, and proliferation of α-smooth muscle cell actin-positive myofibroblasts (23) leading to 20% lumenal occlusion already at 10 d, and nearly total occlusion at 30 d (Figure 1). CsA treatment somewhat delayed the development of epithelial necrosis (Figure 2A). Vehicle in combination with CsA did not inhibit tracheal occlusion compared with vehicle alone (Figures 1 and 2B). CGP 53716 alone and in combination with CsA significantly inhibited the development of OB, compared with vehicle and vehicle in combination with CsA, respectively, but no significant synergistic effect with CGP 53716 and CsA was observed (Figures 1 and 2B).
Fig. 1. Photomicrographs of nontreated syngeneic graft, nontreated allograft, allograft treated with vehicle, allograft treated with CGP 53716, allograft treated with vehicle plus CsA, and allograft treated with CGP 53716 plus CsA 30 d after transplantation. In syngeneic grafts no myofibroproliferation occurred; the airway lumen was covered with normal respiratory epithelium and filled with mucus. In nontreated allografts and allografts treated with vehicle or allografts treated with vehicle plus CsA there was a nearly total occlusion of airway lumen by dense myofibroproliferative tissue. CGP 53716 alone and in combination with CsA significantly ameloriated the development of obliterative lesion. (H&E staining, original magnification, ×40.)
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Fig. 2. The effect of CGP 53716 on (A) epithelial necrosis, (B) the degree of lumenal occlusion, and (C and D) the number of BrdU+ cells in the airway wall (C ) and in myofibroproliferative lesion (D) 10 and 30 d after transplantation. Data are given as means ± SEM. One-factor ANOVA and the Fisher PLSD test were used to determine significance.
[More] [Minimize]The specificity controls of immunostainings showed no immunoreactivity (Figure 3). In normal nontransplanted tracheas, weak PDGF-AA and mild PDGF-BB expression was observed in epithelial and mesenchymal cells while PDGF-Rα and PDGF-Rβ immunoreactivity was nonexistent (Figure 4). In syngeneic grafts, mild PDGF-AA and mild to moderate PDGF-BB expression was localized to recovering epithelium, and, to a lesser extent, to mesenchymal and mononuclear inflammatory cells. Mild PDGF-Rα expression was observed in the epithelium, while PDGF-Rβ expression was nonexistent (Figures 3 and 4). In nontreated allografts, the induction of PDGF-AA was observed at 3 and 10 d and was localized to myofibroproliferative lesion, airway wall SMCs, capillary endothelium and mononuclear inflammatory cells (Figures 3 and 4). The induction of PDGF-Rα immunoreactivity coincided with PDGF-AA expression and was localized mainly to myofibroproliferative lesion, airway wall SMCs, and mononuclear inflammatory cells. In addition, PDGF-Rα expression was detected in allograft epithelium when preserved (Figures 3 and 4). In nontreated allografts, PDGF-BB and PDGF-Rβ expression was mild and mainly localized to airway wall SMCs and mononuclear inflammatory cells, and PDGF-BB was downregulated compared with syngeneic grafts (Figures 3 and 4).
Fig. 3. Photomicrographs of immunoperoxidase staining for PDGF ligand and receptor expression in nontreated syngeneic and allogeneic tracheal grafts 3 d after transplantation. To verify the specificity of immunostaining, primary antibody was preabsorbed with the corresponding peptide antigen and stainings showed no immunoreactivity for PDGF-AA, PDGF-BB, PDGF-Rα, or PDGF-Rβ. In syngeneic grafts, PDGF-AA and PDGF-BB expression was strong in the epithelium, whereas PDGF-Rα and PDGF-Rβ expression was mild. In nontreated allografts, a significant upregulation in PDGF-AA and PDGF-Rα expression was observed in myofibroproliferative lesions and mononuclear inflammatory cells, whereas PDGF-BB and PDGF-Rβ expression remained mild. (Counterstaining with hematoxylin, original magnification ×100, insets ×400.)
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Fig. 4. PDGF ligand and receptor protein expression in nontreated syngeneic grafts and allografts by immunohistochemistry. The gray areas represent the expression levels in normal nontransplanted tracheas, given as means ± SEM. Data are given as means ± SEM. *p < 0.05 and †p < 0.01 by Mann–Whitney U test.
[More] [Minimize]In nontreated allografts, PDGF-A mRNA expression was not significantly altered at the time points investigated, whereas PDGF-B mRNA was slightly increased at 3 d, compared with syngeneic grafts, but remained at nearly the basal level of normal nontransplanted tracheas (Figures 5 and 6). Allograft PDGF-Rα and PDGF-Rβ mRNA expression was significantly induced at 3 d after transplantation, compared to syngeneic grafts (Figures 5 and 6).
Fig. 5. Example of RT-PCR of PDGF ligand and receptor mRNA and GAPDH mRNA transcript levels from a representative analysis that included one cDNA sample from each group, i.e., normal trachea, nontreated syngeneic graft, and allograft 3 d after transplantation. The actual determinations were performed simultaneously, using a 96-well PCR apparatus, and were completed in triplicate.
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Fig. 6. PDGF ligand and receptor mRNA expression in nontreated syngeneic grafts and allografts by semiquantitative RT-PCR. The gray areas represent the expression levels in normal nontransplanted tracheas, given as means ± SEM. Data are given as means ± SEM. *p < 0.05 and †p < 0.01 by Mann–Whitney U test.
[More] [Minimize]In a preliminary study, CGP 53716 induced a slight increase in CsA blood trough levels (Figure 7A). To rule out the possible effects of CsA on the inhibition of OB, CsA doses were determined according to CsA trough levels. For final analysis, 10 rats per group with matched CsA trough levels were chosen (Figure 7B) and only these grafts were used for histological and immunohistochemical analysis.
Fig. 7. Effect of CGP 53716 on CsA blood trough levels. (A) A preliminary study revealed that CGP 53716 induced a slight increase in CsA blood levels. (B) For the final histological and immunohistochemical analysis the CsA blood levels were matched between CGP 53716-treated and nontreated rats. The gray area represents the aimed CsA blood trough level, according to which CsA dosing was adjusted. *p < 0.05 by Mann–Whitney U test.
[More] [Minimize]CGP 53716 treatment had no effect on airway wall inflammatory cell proliferation either in nonimmunosuppressed or immunosuppressed allografts (Figure 2C). On the other hand, CGP 53716 significantly inhibited the proliferation of myofibroblasts at 10 d, when compared with the vehicle group (Figure 2D). A combination of CsA [(1 mg/kg)/d] with CGP 53716 did not further inhibit myofibroproliferation at 10 d, but significantly reduced myofibroproliferation at 30 d, when compared with the vehicle plus CsA group (Figure 2D).
In all groups epithelial MHC class II expression was strong at 10 d, subsiding thereafter. A moderate airway wall infiltration of CD4+ and CD8+ T cells and to a lesser extent of ED3+ activated macrophages at 10 and 30 d after transplantation was observed (Table 1). Many of the infiltrating inflammatory cells expressed IL-2R and MHC class II (Table 1). Treatment with either CsA or CGP 53716 did not significantly affect the intensity or immune activation of the airway wall inflammation (Table 1).
| Time after Transplantation | Group | n | ED3+Macrophages | CD4+ T cells (W3/25) | CD8+ T cells (OX8) | IL-22R (CD25) | MHC Class II (OX6) | |||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 10 d | Vehicle | 10 | 0.3 ± 0.1 | 1.5 ± 0.1 | 1.4 ± 0.1 | 1.1 ± 0.1 | 1.9 ± 0.1 | |||||||
| CGP 53716 | 10 | 0.2 ± 0.0 | 1.3 ± 0.2 | 1.3 ± 0.1 | 0.8 ± 0.1 | 1.5 ± 0.1 | ||||||||
| Vehicle + CsA | 10 | 0.3 ± 0.1 | 0.9 ± 0.1 | 1.6 ± 0.1 | 0.7 ± 0.1 | 1.7 ± 0.1 | ||||||||
| CGP 53716 + CsA | 10 | 0.3 ± 0.1 | 0.9 ± 0.1 | 1.3 ± 0.2 | 0.8 ± 0.1 | 2.1 ± 0.1 | ||||||||
| 30 d | Vehicle | 10 | 0.6 ± 0.1 | 1.2 ± 0.1 | 1.2 ± 0.1 | 0.6 ± 0.1 | 2.1 ± 0.2 | |||||||
| CGP 53716 | 10 | 0.3 ± 0.1 | 1.1 ± 0.2 | 1.2 ± 0.1 | 0.6 ± 0.1 | 1.4 ± 0.2 | ||||||||
| Vehicle + CsA | 10 | 0.8 ± 0.1 | 1.2 ± 0.1 | 1.5 ± 0.1 | 0.9 ± 0.2 | 2.4 ± 0.1 | ||||||||
| CGP 53716 + CsA | 10 | 0.5 ± 0.1 | 1.4 ± 0.1 | 1.5 ± 0.1 | 0.8 ± 0.2 | 2.0 ± 0.2 |
This study indicates a pivotal role for PDGF, especially PDGF-AA, and for PDGF-Rα, in the pathogenesis of OB, a manifestation of chronic rejection. We found an increase in PDGF-Rα and -Rβ mRNA expression, and in PDGF-AA and -Rα immunoreactivity, during the development of experimental OB. In addition, inhibition of PDGF-R significantly reduced myofibroproliferation and airway occlusion, suggesting a regulatory role for PDGF in OB.
Although several studies indicate a role for PDGF in the pathogenesis of fibroproliferative lung disease and chronic rejection (15-20), the present study suggests a particular role for PDGF-AA and -Rα in the pathogenesis of experimental OB. IL-1 and TNF-α, also upregulated during OB development (23, 24), induce PDGF-AA and -Rα expression leading to proliferation of lung myofibroblasts (27, 28). Accordingly, in this study upregulation of PDGF-AA and -Rα occurred concomitantly with the peak of inflammation and myofibroproliferation at 3 and 10 d, and subsided within 30 d with downregulation of proinflammatory cytokines and myofibroproliferation (23, 24). The slight increase in PDGF-Rβ protein expression at 30 d, may be due to increased number of mesenchymal cells observed at this time point.
A specific role for PDGF-AA and -Rα, and posttranscriptional regulation of PDGF-A mRNA, has been suggested in chronic heart allograft rejection (20, 29). Alternative mRNA splicing of PDGF-A chain results in two functionally different A-chain precursors: the long A chain containing exon 6 and the short A chain lacking exon 6 (30). Cytokine-activated cells produce the long chain, whereas resting cells express the short chain of PDGF-A (8, 29), and the long-chain PDGF-AA is required for full activation of PDGF-Rα protein-tyrosine kinase (31). In cardiac allografts only the long PDGF-A chain is produced, resulting in the induction of PDGF-Rα expression, not seen in normal hearts (20, 29). Although the primers and antibodies used in this study do not differentiate between the long and short chains of PDGF-A, we hypothesize that the proliferative responses during OB development are mediated by the long-chain PDGF-AA and PDGF-Rα, whereas syngeneic grafts produce predominantly short-chain PDGF-AA and PDGF-BB, and PDGF-Rα expression remains at the basal level of nontransplanted tracheas.
There was some discrepancy between PDGF mRNA and protein expression, which may in part be due to the limitations of the semiquantitative RT-PCR method. It does not distinquish minor differences between the groups studied or localize the expression, which may be of pathophysiological significance. PDGF-AA protein expression was significantly induced, while mRNA expression remained at the level of syngeneic grafts. Strong PDGF-AA expression was observed in allograft myofibroblasts and inflammatory cells, but also in the syngeneic graft epithelium, and the total intragraft mRNA remained the same although different cells produced the protein. In addition, posttranscriptional regulation, including splicing (32), may explain some of the differences in mRNA and protein expression. PDGF-Rα protein expression was induced throughout the 30-d period, whereas mRNA was induced only at 3 d, indicating that PDGF-Rα protein may be longer lived than its mRNA. PDGF-BB protein expression was reduced in allografts, whereas its mRNA expression was slightly induced at 3 d. However, mRNA and protein expression in both groups remained nearly at the level of nontransplanted controls and the different distribution of PDGF-BB-expressing cells may have affected these results. PDGF-Rβ mRNA expression was induced in allografts at 3 d, whereas PDGF-Rβ protein expression was mild, and only slight upregulation in immunoreactivity could be demonstrated.
Our second objective was to study whether OB may be inhibited by a PDGF-R protein-tyrosine kinase inhibitor, CGP 53716. CGP 53716 was given at a dosage of 50 (mg/kg)/d; 100 (mg/kg)/d has been shown to yield CGP 53716 plasma levels of 2 μM in rats (E. Buchdunger, personal communication, 1998). The 50% inhibitory concentration (IC50) for inhibition of PDGF-R by CGP 53716 is approximately 0.1 μM, whereas the IC50 for other protein kinases is 10–500 μM, demonstrating high selectivity for PDGF-R tyrosine kinase (21), and indicating that the effects of CGP 53716 seen in this study were due to specific inhibition of PDGF-R tyrosine kinase. The concentration of CGP 53716 used is more effective in inhibiting PDGF-Rα than PDGF-Rβ in vitro (33), also supporting the role of the PDGF-AA–PDGF-Rα pathway in the development of OB.
We found that CGP 53716 without any additional immunosuppression markedly inhibited myofibroproliferation and airway occlusion. CGP 53716 had no effect on inflammatory cell infiltration and proliferation, or on the intensity of immune activation, indicating that CGP 53716 is not immunosuppressive per se, but rather that its effects are mediated via inhibition of myofibroproliferation. Thus, the inhibitory effects seen here are most likely due to a specific inhibition of PDGF-R protein-tyrosine kinase activity. However, the inhibition of OB by CGP 53716 was not complete, indicating that other mitogens operate in this disease process. No synergistic effect with CsA was observed.
In conclusion, this study suggests a regulatory role for PDGF, especially for PDGF-AA and -Rα, in the development of OB, a manifestation of chronic lung allograft rejection, and demonstrates that inhibition of PDGF-R protein-tyrosine kinase activation markedly reduces OB. Thus, receptor protein-tyrosine kinase inhibitors may provide a novel therapeutic strategy for the prevention of chronic rejection.
The authors acknowledge the excellent technical assistance of E. Aaltola, R.N., and M. Anttila, R.N.
Supported by the Jalmari and Rauha Ahokas Foundation, the Finnish Medical Society Duodecim, the University of Helsinki, Helsinki University Central Hospital Research Funds, the Finnish Foundation of Cardiovascular Research (Helsinki, Finland), the Leiras Research Foundation and Farmos Pharmaceuticals (Turku, Finland), as well as Contract No. BMH-4CT95-1160 of Biomed 2, from the European Union.
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