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Published ahead of print on March 18, 2005, doi:10.1164/rccm.200408-1001OC
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American Journal of Respiratory and Critical Care Medicine Vol 171. pp. 1421-1429, (2005)
© 2005 American Thoracic Society
doi: 10.1164/rccm.200408-1001OC

Original Article

Dual Role of Vascular Endothelial Growth Factor in Experimental Obliterative Bronchiolitis

Rainer Krebs, Jussi M. Tikkanen, Antti I. Nykänen, Jeanette Wood, Michael Jeltsch, Seppo Ylä-Herttuala, Petri K. Koskinen and Karl B. Lemström

Cardiopulmonary Research Group, Transplantation Laboratory, University of Helsinki/Helsinki University Central Hospital; Molecular Cancer Biology Laboratory, Biomedicum Helsinki, University of Helsinki; Division of Nephrology, Department of Medicine, and Department of Cardiothoracic Surgery, Helsinki University Central Hospital, Helsinki; A.I. Virtanen Institute for Molecular Sciences, University of Kuopio, Kuopio, Finland; and Novartis Pharma, Basel, Switzerland

Correspondence and requests for reprints should be addressed to Karl Lemström, M.D., Ph.D., Transplantation Laboratory, P.O. Box 21 (Haartmaninkatu 3), FIN-00014 Helsinki, Finland. E-mail: karl.lemstrom{at}helsinki.fi


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Obliterative bronchiolitis (OB) is the major limitation for long-term survival of lung allograft recipients. We investigated the role of vascular endothelial growth factor (VEGF) in the development of OB in rat tracheal allografts. In nonimmunosuppressed allografts, VEGF mRNA and protein expression vanished in the epithelium and increased in smooth muscle cells and mononuclear inflammatory cells with progressive loss of epithelium and airway occlusion compared with syngeneic grafts. Intragraft VEGF overexpression by adenoviral transfer of a mouse VEGF164 gene increased early epithelial cell proliferation and regeneration but increased microvascular remodeling and lymphangiogenesis and luminal occlusion by more than 50% compared with AdlacZ-treated allografts. Although VEGF receptor inhibition decreased early epithelial regeneration in noninfected allografts, it reduced microvascular remodeling, lymphangiogenesis, intragraft traffic of CD4+ and CD8+ T cells, and the degree of luminal occlusion. Simultaneous VEGF gene transfer and platelet-derived growth factor receptor inhibition with imatinib preserved respiratory epithelium and totally prevented luminal occlusion. In conclusion, our findings indicate that VEGF has a dual role in transplant OB. Our results suggest that VEGF may protect epithelial integrity. On the other hand, VEGF may enhance luminal occlusion by increasing the recruitment of mononuclear inflammatory cells with platelet-derived growth factor acting as a final effector molecule in this process.

Key Words: angiogenic growth factors • lung • transplantation

Lung transplantation is the only method currently available to return patients with end-stage pulmonary disease to normal life. Bronchiolitis obliterans syndrome is the leading cause of mortality after lung transplantation and the reason why the 10-year patient survival is only 20% (1). Histologically, bronchiolitis obliterans syndrome presents as obliterative bronchiolitis (OB) (2), a pulmonary manifestation of chronic rejection. The pathogenetic mechanisms leading to OB remain largely unknown and no specific treatment for OB is available. The development of OB is characterized by features of dysregulated repair and may be divided into two phases: persistent alloimmune injury, directed especially at the bronchiolar epithelium, followed by a chronic fibroproliferative reparative process leading to gradual occlusion of the airway lumen (3).

Angiogenesis, lymphangiogenesis, and microvascular remodeling are recognized features of chronic inflammatory diseases and therefore may play a pivotal role in chronic lung inflammatory processes, such as asthma and chronic bronchitis (4, 5). Formation of new vessels and remodeling of existing ones are likely to be induced by multiple growth factors. Vascular endothelial growth factor (VEGF) is a potent angiogenic factor whose activities include endothelial cell survival, proliferation, and migration (6). VEGF acts as a proinflammatory cytokine by increasing endothelial permeability and inducing expression of endothelial adhesion molecules that bind leukocytes (7, 8). It is also a chemoattractant to monocytes (9). Although VEGF has traditionally been considered as an endothelial cell–specific growth factor, recent reports suggest that it may be an important growth factor for epithelial cells, too (10). Furthermore, a recent study shows that VEGF induces remodeling and enhances TH2-mediated sensitization and inflammation in the lung (11).

Angiogenesis, lymphangiogenesis, and vascular remodeling may account for the failure of conventional immunosuppressive drugs to prevent the development of OB. Because VEGF is central in angiogenesis and to a lesser extent also in lymphangiogenesis, we investigated whether VEGF ligand and receptors are induced during experimental OB in rat tracheal allografts, and studied the biological role and mechanisms of action of VEGF in this process. Here, we demonstrate that the localization of VEGF protein and mRNA expression is switched from epithelial expression in normal trachea and syngeneic tracheal grafts to airway smooth muscle cells (SMCs) and mononuclear inflammatory cells in tracheal allografts. Furthermore, adenoviral VEGF gene transfer accelerates the development of OB, whereas blocking of VEGF receptor (VEGFR) tyrosine kinase activity inhibits it, suggesting an important role for VEGF in this process. Although VEGF seems to promote epithelial regeneration after early alloimmune injury, we show that it is associated with enhanced alloimmune activation, lymphangiogenesis, microvascular remodeling, and the development of OB. Finally, we show that the proproliferative effects of VEGF are mediated via platelet-derived growth factor (PDGF), because simultaneous VEGF gene transfer and PDGF receptor (PDGFR) tyrosine kinase inhibition result in complete prevention of OB.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Heterotopic Rat Tracheal Transplantation
Specific pathogen-free inbred male Dark Agouti (DA; AG-B4, RT1a) and Wistar Furth (WF; AG-B2, RT1u) rats (Harlan, Horst, The Netherlands) weighing 200 to 300 g and 2 to 3 months old were used as described. All transplantations were performed heterotopically to the recipient's bursa omentalis as described (12). Syngeneic tracheal grafts were transplanted from DA donors to DA recipients and allografts from DA donors to WF recipients. All grafts were harvested 10 and 30 days after transplantation. Nontransplanted DA trachea served as normal controls. Permission for animal experimentation was obtained from the State Provincial Office of Southern Finland. All rats received humane care in compliance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (National Academy Press, Washington, DC, 1996).

Experimental Design
First, we investigated VEGF ligand and VEGFR expression during the development of OB in nonimmunosuppressed rats at mRNA and protein level by in situ hybridization and immunohistochemistry, respectively (for details, see online supplement). Second, we performed gene transfer in tracheal allografts using adenoviruses encoding mouse VEGF164 (AdVEGF) or ß-galactosidase (AdlacZ) and transplanted these allografts into recipients receiving cyclosporine A (CsA) 1.5 mg/kg/day to investigate whether VEGF enhances the development of OB. To investigate the efficiency of the adenoviral gene transfer, we infected DA tracheal grafts with ß-galactosidase–encoding adenovirus for whole-mount X-gal staining and with enhanced green fluorescent protein (EGFP)–encoding adenovirus (AdEGFP) for fluorescent microscopy analysis. The expression was analyzed after 3 and 7 days (both groups) and 10 and 30 days (AdlacZ-infected grafts only; for details, see online supplement and Reference 13). Concomitantly with VEGF gene transfer, one group of recipients received VEGFR tyrosine kinase inhibitor PTK787/ZK222584 (PTK787; PTK; Novartis, Basel, Switzerland), a second group received a PDGFR tyrosine kinase inhibitor (imatinib), and a third group received N-nitro-L-arginine methyl ester (L-NAME) for the whole study period to investigate the mechanisms how VEGF regulates epithelial regeneration (L-NAME) and the development of OB (PTK787 and imatinib). Third, we gave allograft recipients PTK787 or vehicle and CsA 1 mg/kg/day to investigate the effect of inhibition of VEGFR activation on the development of OB. All analyses were performed by two independent observers in a double-blinded manner with excellent interobserver correlation.

Drug Regimens
CsA (Novartis) was administered subcutaneously at the doses described previously. PTK787 (Novartis) was given 100 mg/kg/day via an orogastric tube. Imatinib (Novartis) was administered 10 mg/kg/day intraperitoneally (for details, see online supplement). L-NAME (Sigma, St. Louis, MO) was administered into drinking water 1 g/L for effective inducible nitric oxide (NO) synthase inhibition (14). Neither PTK nor imatinib altered CsA 24-hour blood-trough levels in our preliminary study.

Microvascular Remodeling and Lymphangiogenesis
Allograft vascularization and lymphangiogenesis were determined using immunohistochemistry (for details, see online supplement). Mouse antirat endothelial cell antigen-1 (RECA-1, 1:10; No. MCA970; Serotec, Oxford, UK) was used to quantitate the number of allograft blood vessels, mouse anti–high-molecular-weight melanoma-associated antigen (MAb 225.28; 1:50; a generous gift from Dr. S. Ferrone, Roswell Park Cancer Institute, Buffalo, NY) for detection of activated pericytes (15), and rabbit anti–LYVE-1 (recognizing a receptor for hyaluronan in lymphatic endothelium; 1:1000) for detection of lymphatic vessels. For amplification of the LYVE-1 immunohistochemical signal, the TSA Biotin system (Perkin-Elmer, Boston, MA) was used. The results are expressed as positive vessels/allograft tracheal cross-section.

Statistical Analyses
All data are expressed as mean ± SEM. Student's t test and analysis of variance were used for parametric comparisons, whereas Mann-Whitney and Kruskal-Wallis and Dunn tests were used for nonparametric comparisons (StatView 4.1 program; Abacus Concepts, Inc., Berkeley, CA). A p value of less than 0.05 was regarded as statistically significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of VEGF Ligand and VEGFR during the Development of OB
Using our heterotopic rat tracheal allograft model, we investigated VEGF ligand and VEGFR expression during the development of OB in nonimmunosuppressed normal DA trachea, tracheal syngeneic grafts (DA to DA), and allografts (DA to WF) at mRNA and protein level by in situ hybridization and immunohistochemistry, respectively. This permitted us to distinguish between changes in VEGF ligand and VEGFR expression induced by the transplantation procedure itself (normal vs. syngeneic grafts) and the changes caused by the alloimmune response (syngeneic grafts vs. allografts). In nonimmunosuppressed syngeneic grafts, the epithelium had nearly recovered from ischemic injury at 10 days and no myofibroproliferation was seen at 30 days. In nonimmunosuppressed allografts, there was progressive loss of epithelium seen by 10 days and intense myofibroproliferation nearly totally occluding the lumen at 30 days (Figure 1A, insets).



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  		Figure 1. Intragraft vascular endothelial growth factor (VEGF), VEGF receptor 1 (VEGFR-1), and VEGFR-2 immunoreactivity in normal Dark Agouti (DA) tracheas, syngeneic grafts, and allografts 10 (photomicrographs) and 30 days after transplantation. Heterotopic tracheal transplantations were performed to investigate intragraft VEGF, VEGFR-1, and VEGFR-2 immunoreactivity in nonimmunosuppressed recipients. Epithelial expression and the surrounding airway wall were scored separately to compare the localization of expression of VEGF and its receptors between normal, syngeneic, and allogeneic groups. This permitted us to distinguish between changes in VEGF ligand and VEGFR expression induced by the transplantation procedure itself (normal vs. syngeneic grafts) and the changes caused by the alloimmune response (syngeneic grafts vs. allografts). (A, insets) After transplantation, syngeneic grafts sustain normal ciliated respiratory epithelium and show no occlusion 30 days after transplantation in contrast to nonimmunosuppressed allografts that undergo progressive loss of epithelium and are completely occluded by a myofibroproliferative lesion by 1 month. (A, B) In normal DA tracheas and syngeneic grafts, VEGF expression was localized to the ciliated respiratory epithelium and myofibroblast-like cells of the airway wall, whereas in nonimmunosuppressed allografts, VEGF expression was faint in the damaged epithelium, but intense in the smooth muscle cell (SMC) layer (not shown) and mononuclear inflammatory cells of the airway wall. (C–F) VEGFR-1 and VEGFR-2 immunoreactivities were observed in ciliated respiratory epithelial cells of normal DA trachea and regenerating epithelial cells of syngeneic grafts. (C, D) The injured allograft epithelium did not express VEGFR-1, but faint staining was observed in mononuclear inflammatory cells. (E, F) Allograft VEGFR-2 expression was observed in epithelial cells and airway wall mononuclear cells. Sections were developed by biotin-avidin system (red) and counterstained with hematoxylin (blue). Incubation of primary antibodies with specific control peptides before staining resulted in the absence of immunoreactivity (insets). The immunoreactivity was scored from nonexistent (0) to strong (3). For insets in A, histologic sections were stained with Masson trichrome. Data are mean ± SEM (n = 6–8/group). *p < 0.05, **p < 0.01, {dagger}p < 0.005 by Mann-Whitney U test, compared with syngeneic grafts. Scale bar represents 50 µm.

 
Immunohistochemical analysis of normal nontransplanted trachea showed strong VEGF expression localized to respiratory epithelium and faint expression to the microvasculature of the airway wall. In syngeneic grafts, moderate VEGF expression was seen in the epithelium and in the microvasculature of the airway wall 10 and 30 days after transplantation. In nonimmunosuppressed allografts, the severely damaged epithelium showed decreased VEGF immunoreactivity, whereas intense VEGF expression was observed in SMCs and infiltrating mononuclear inflammatory cells and in the microvasculature of the airway wall at 10 days. At 30 days, when the alloimmune reaction already started to subside, moderate VEGF expression was noted in the airway wall and faint expression in the myofibroproliferative lesion (Figures 1A and 1B).

Moderate epithelial VEGFR-1 expression was observed in normal trachea. In syngeneic grafts, mild epithelial and microvascular endothelial VEGFR-1 expression could be detected at 10 or 30 days. In nonimmunosuppressed allografts, weak VEGFR-1 expression was observed in the microvascular endothelium of the airway wall and graft-infiltrating mononuclear cells (Figures 1C and 1D).

Faint epithelial VEGFR-2 expression occurred in epithelial cells of normal and syngeneic grafts. In nonimmunosuppressed allografts at 10 days, mild VEGFR-2 was found in the epithelium and moderate expression in the microvasculature of the airway wall (Figures 1E and 1F).

In general, VEGF ligand and VEGFR mRNA expression correlated with that of protein expression (Figure 2). VEGF mRNA expression was localized to epithelial cells of normal and syngeneic trachea, whereas allografts expressed VEGF mRNA mainly in SMCs and mononuclear inflammatory cells. The epithelium of normal and syngeneic trachea expressed VEGFR-1, whereas moderate VEGFR-1 expression was observed in the allograft airway wall. VEGFR-2 expression was mild in all groups and localized mainly to epithelial cells and airway wall blood vessels.



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  		Figure 2. Heterotopic tracheal transplantation was performed to investigate intragraft VEGF, VEGFR-1, and VEGFR-2 mRNA expression. No treatment was administered. Intragraft (A, B) VEGF, (C, D) VEGFR-1, and (E, F) VEGFR-2 mRNA expression, analyzed by in situ hybridization in normal DA trachea, syngeneic graft, and allograft epithelium and airway wall 10 days after transplantation. Sections were hybridized with digoxigenin-labeled probes and the reaction was revealed by alkaline phosphatase-conjugated antidigoxigenin antibody (blue). Sections were counterstained with nuclear fast red. Sense controls did not show reactivity (insets). Scale bar represents 50 µm.

 
Adenoviral Gene Transfer Leads to a Transient Local Gene Expression Localized Mainly to the Allograft Epithelium
We have previously shown that VEGF is proinflammatory and increases chronic rejection in rat cardiac allografts (16). This led us to hypothesize that VEGF might also enhance the development of OB, a manifestation of chronic rejection in lung allografts. Base immunosuppression with 1.5 mg/kg/day of CsA leads to tracheal occlusion of 29 ± 10% at 1 month after transplantation (12). We calculated that the possible deleterious effect of VEGF overexpression would become apparent with this dose.

To characterize the biology of adenoviral infection and gene transfer in our model, we performed additional lacZ- and EGFP-encoding adenoviral constructs. Peak infectivity was seen at 3 days when epithelium showed strong focal ß-galactosidase and EGFP-expression (Figures 3B, 3D, and 3F). At 7 days, the level of infection was already reduced although mild epithelial ß-galactosidase and EGFP expression was observed (data not shown). Unfortunately, because of antibody cross-reactivity, we could not reliably distinguish mouse VEGF164 from endogenous rat VEGF and determine the rate of adenoviral VEGF expression, but we observed increased VEGF expression in the allograft epithelium at 10 days in the AdVEGF-infected group (Figure 3H). No ß-galactosidase activity nor EGFP fluorescent signal was observed from the liver or the surrounding omentum at any time point (data not shown).



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  		Figure 3. The biology of adenoviral infection in rat heterotopic tracheal allografts in recipients receiving cyclosporine A (CsA) 1.5 mg/kg/day. After the donor trachea was harvested, it was incubated with 0.2 x 109 plaque-forming units of adenoviral vector encoding either mouse VEGF164 (AdVEGF) or ß-galactosidase (AdlacZ) at +4°C for 30 minutes. Ethylenediaminetetraacetic acid was used to permeabilize the epithelium. After incubation, the grafts were transplanted into the recipient's bursa omentalis. First, using whole mount X-gal staining, we analyzed intragraft ß-galactosidase expression after AdlacZ gene transfer at 3, 7, 10, and 30 days after transplantation and adenoviral infection. Compared with noninfected controls (A), the epithelial cells of infected grafts showed intense ß-galactosidase activity at 3 days (B). Only a few X-gal–positive cells were seen in the airway wall, suggesting that the infection was mainly localized to epithelial cells. The activity subsided thereafter, with slight expression evident at 7 days and nearly no expression visible at 10 and 30 days (data not shown). To confirm our results, we performed a second experiment using enhanced green fluorescent protein–encoding adenovirus (AdEGFP) gene transfer. At 3 days, AdEGFP-infected tracheal allografts showed epithelial cell expression scattered widely throughout the inner surface (i.e., epithelium) of the tracheal allografts (D). (F) An en face view of the allograft epithelium shows intense EGFP-expression at 3 days, fading away at later time points (data not shown). Noninfected controls showed no specific fluorescent signal (C, E). Using immunohistochemistry, we analyzed intragraft VEGF expression after AdlacZ and AdVEGF gene transfer at 10 days after transplantation and adenoviral infection. Compared with AdlacZ-infected tracheas (G), AdVEGF-infected tracheas showed increased levels of VEGF expression in the epithelium (H) and airway wall (insets of G, H). Sections of whole mount X-gal stainings were counterstained with nuclear fast red. Immunohistochemical sections were counterstained with hematoxylin. Fluorescence microscopy original magnification, 40x (C, D) and 200x (E, F). The arrows (C, D) indicate the tracheal epithelium. Scale bars represent 50 µm.

 
VEGF Gene Transfer Increases Early Epithelial Regeneration but Enhances the Development of OB
AdlacZ gene transfer did not induce OB formation compared with our historical noninfected allografts because the level of occlusion was 17 ± 2% compared with the 29 ± 10% occlusion rate in our previous series (12), suggesting that the adenovirus infection itself does not promote the development of OB.

AdVEGF gene transfer induced epithelial cell proliferation (Ki67+ cells, 113 ± 22 vs. 56 ± 14 T cells in AdlacZ group; p < 0.05), leading to a 50% reduction in epithelial loss at 10 days (p = nonsignificant). However, it tripled airway occlusion at 30 days compared with AdlacZ-immersed allografts (p < 0.05; Figure 4). Simultaneous treatment with PTK787 negated the deleterious effect of VEGF gene transfer on tracheal allograft obliteration at 30 days (Figure 5).



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  		Figure 4. We performed adenovirus-mediated mouse VEGF164 or lacZ gene transfer in tracheal allografts and transplanted these allografts into recipients receiving CsA 1.5 mg/kg/day to investigate whether VEGF enhances the development of obliterative bronchiolitis (OB). As depicted in Figure 3, this led to a transient epithelial overexpression of VEGF that peaked at 3 days after transplantation. Shown here are the effects of AdlacZ or AdVEGF gene transfer on (A) epithelial necrosis and (B) luminal occlusion at 10 and 30 days, on (C) the number of Ki-67+ proliferating epithelial cells, and on (D–F) allograft inflammation at 10 days. (A) Although AdVEGF gene transfer halved epithelial necrosis at 10 days, both groups showed a nearly total loss of epithelium at 30 days. (B) AdVEGF gene transfer tripled the development of luminal occlusion at 30 days compared with AdlacZ-treated controls. (C) AdVEGF gene transfer induced epithelial cell proliferation at 10 days (Ki67+ cells) but did not affect the number of graft-infiltrating (D) ED1+ macrophages, (E) CD4+ T cells, or (F) CD8+ T cells, when compared with AdlacZ-treated allografts. Histologic sections were stained with hematoxylin–eosin, and immunohistochemical sections were developed by biotin-avidin system (red) and counterstained with hematoxylin (blue). Specificity controls with IgG subfractions did not show immunoreactivity (insets). Data are mean ± SEM (n = 7–10/group). *p < 0.05 by Student's t test, compared with AdlacZ-treated allografts. Scale bars represent 50 µm. EL = epithelial layer; L = lumen; SEL = subepithelial layer.

 


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  		Figure 5. VEGF induces the development of OB indirectly through activation of the platelet-derived growth factor (PDGF) pathway. (A; E, blue bar) In AdlacZ-treated controls receiving background CsA immunosuppression, the ciliated respiratory epithelium has changed into squamous epithelium (inset) but the tracheal lumen remains nearly unoccluded. (B; E, red bar) AdVEGF gene transfer enhances epithelial damage (inset) and the development of OB at 30 days, and (C; E, green bar) this effect is prevented by selective VEGFR tyrosine kinase inhibition with PTK787, suggesting an important role for VEGF in regulating the development of OB. (D; E, black bar) The effect of AdVEGF gene transfer is mediated by PDGFR, because simultaneous treatment with imatinib, a selective protein kinase inhibitor for PDGFR, completely preserved respiratory epithelium (inset) and abolished luminal occlusion at 30 days. Sections were stained with hematoxylin–eosin. Data are mean ± SEM (n = 7–10/group). {dagger}p < 0.005 by analysis of variance with Bonferroni correction, when compared with AdVEGF-immersed allografts. Scale bar represents 50 µm.

 
VEGF Enhances the Regeneration of Respiratory Epithelium Independently of NO and Accelerates the Development of OB Downstream of PDGFR
VEGF is known to induce NO production and, in turn, NO is believed to have a protective effect on airway epithelium after experimental transplantation (17, 18). To establish whether the early protective effect of VEGF on tracheal epithelium was mediated via increased NO production, we treated AdVEGF-immersed tracheal allograft recipients with NO synthase inhibitor L-NAME. No effect on epithelial necrosis was observed 10 days after transplantation, when compared with AdVEGF-treated allografts (15 ± 7.6 vs. 13.5 ± 3.6%, p = nonsignificant). We also treated AdVEGF-infected allograft recipients with PDGFR tyrosine kinase inhibitor (imatinib) to evaluate whether VEGF overexpression accelerates the development of OB via PDGF, which is known to have proproliferative and proobliterative properties (19). Concomitant AdVEGF infection and imatinib treatment preserved respiratory epithelium and totally abolished luminal occlusion at 30 days, when compared with AdVEGF-treated allografts (p < 0.005; Figure 5).

VEGFR Inhibition Decreases Early Epithelial Regeneration but Reduces Luminal Occlusion
Allograft recipients were given PTK787 or vehicle and CsA 1 mg/kg/day to investigate the effect of inhibition of VEGFR activation on the development of OB. Treatment with PTK787 did not significantly affect epithelial necrosis at 10 days (p = nonsignificant) but markedly inhibited epithelial cell proliferation (Ki67+ cells, 17 ± 5 vs. 110 ± 18 T cells in vehicle-treated group; p < 0.0005). It reduced epithelial necrosis (p < 0.05) and tracheal allograft occlusion (p < 0.005) by over 50% at 30 days, when compared with vehicle-treated controls (Figure 6).



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  		Figure 6. Effect of vehicle or VEGFR protein tyrosine kinase inhibition with PTK787 treatment on (A) epithelial necrosis and (B) luminal occlusion at 10 and 30 days, on (C) the number of Ki-67+ proliferating epithelial cells, and on (D–F) allograft inflammation at 10 days. CsA was administered as background immunosuppression 1.0 mg/kg/day. (A, C) Although VEGFR inhibition by PTK787 reduced the number of Ki-67+ proliferating epithelial cells at 10 days with a trend toward increased epithelial necrosis, it reduced (A) epithelial necrosis and (B) tracheal allograft occlusion at 30 days and (D–E) the number of graft-infiltrating CD4+ and CD8+ T cells at 10 days. Histologic sections were stained with hematoxylin–eosin; immunohistochemical sections were developed by biotin-avidin system (red) and counterstained with hematoxylin (blue). Data are mean ± SEM (n = 8–10/group). *p < 0.05, {dagger}p < 0.005, {ddagger}p < 0.001, §p < 0.0005 by Student's t test compared with vehicle-treated allografts. Scale bars represent 50 µm. C = cartilage; EL = epithelial layer; L = lumen; SEL = subepithelial layer.

 
VEGFR Inhibition Reduces Allo-specific T-Cell Infiltration
We analyzed allograft inflammation at the time of peak inflammatory response 10 days after transplantation (12). AdVEGF gene transfer had no significant effect on the number of graft-infiltrating CD4+ T cells, CD8+ T cells, or ED1+ macrophages compared with AdlacZ-treated allografts (Figure 4). On the other hand, treatment with PTK787 significantly reduced the number of graft-infiltrating CD4+ and CD8+ T cells (p < 0.005 and p < 0.001, respectively) compared with vehicle-treated allografts (Figure 6).

Effect of VEGF on Microvascular Remodeling and Lymphangiogenesis
Because this is a nonvascularized model, formation of blood and lymphatic vessel supply to the graft is required after transplantation. All allografts developed a strong peritracheal blood supply by 10 days as demonstrated by RECA-1+ immunohistochemistry for vascular endothelium (Figure 7). The different treatment regimens had no effect on the number of RECA-1+ blood vessels. However, VEGF gene transfer increased and PTK787 treatment decreased the number of remodeling blood vessels as shown by high-molecular-weight melanoma-associated antigen–positive immunostaining for activated pericytes (p < 0.05) and the number of LYVE-1+ lymphatic vessels, when compared with appropriate controls, respectively (Figure 7).



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  		Figure 7. Effect of AdVEGF gene transfer and PTK787 treatment on the number of vessels per tracheal cross-section staining positively for (A) rat endothelial cell antigen (RECA-1) for vascular and lymphatic endothelium, (B) for high-molecular-weight melanoma-associated antigen (HMW-MAA) for activated pericytes/SMCs, and (C) for lymphatic vessel hyaluronic acid receptor (LYVE-1) for lymphatic endothelium. Sections were developed by biotin-avidin system (red) and counterstained with hematoxylin (blue). Data are mean ± SEM (n = 6–10/group). *p < 0.05 by Student's t test, when compared with vehicle-treated allografts. Scale bar represents 50 µm.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Heterotopic tracheal allografts develop myofibroproliferative lesions akin to those found in small and large airways of human lung allografts with OB (20). Early in the development of OB, the airway epithelium prominently expresses major histocompatibility complex class II, enabling direct presentation of foreign antigens to alloreactive T cells. In addition, graft-infiltrating recipient antigen-presenting cells may process donor antigens for indirect B- and T-cell activation. The recruitment of CD4+ and CD8+ T cells and macrophages into the airway wall induces local cytokine and growth factor production leading to epithelial injury, microvascular remodeling, and migration and proliferation of {alpha}-SMC actin–positive cells, and gradual occlusion of the airway lumen (19, 21). In syngeneic grafts lacking alloimmune-mediated injury, ischemia-reperfusion injury alone does not induce the development of OB, underlining the central role of the alloimmune response in this disease process (21).

VEGF is a proangiogenic and proinflammatory growth factor that is highly expressed in pulmonary epithelial cells and is an important growth factor for these cells (10). After lung transplantation, ischemia-reperfusion injury and thereby reduced VEGF protein expression leads to alveolar epithelial damage in a rat model (22). In bronchoalveolar lavage fluid of lung transplant recipients, the concentrations of VEGF165 were significantly reduced at early time points but increased with time in the absence of significant rejection or infection. The mean VEGF concentration remained significantly lower if bronchiolitis obliterans syndrome was present at 6 months after transplantation (23). In this study, intense VEGF immunoreactivity was localized in the respiratory epithelium in normal trachea and syngeneic grafts, suggesting that epithelium may be the main source of VEGF in the absence of rejection. In untreated allografts, VEGF mRNA and protein expression decreased in the epithelium together with progressive epithelial injury but increased in SMCs and mononuclear inflammatory cells during alloimmune activation. Thus, it seems that, in stable allografts, epithelial cells may secrete VEGF to bronchoalveolar lavage fluid, and epithelial injury leads to a reduction in this secretion. VEGF expression in mononuclear inflammatory cells in tracheal allografts agrees with the observations in cardiac allografts during acute and chronic rejection where VEGF immunoreactivity positively correlated with rejection (16, 24). In the alloimmune environment, the localization but not the intensity of VEGF expression was altered and may explain in part the shift from the protective to deleterious role (25).

Although adenovirus-mediated gene transfer led to only short-lived overexpression of VEGF, VEGF gene transfer has long-lasting effects on tissue remodeling, as suggested by a recent study (11). The brief adenovirus-mediated VEGF expression alleviated epithelial loss, resulting in increased endogenous VEGF expression at 10 days. In addition, the proinflammatory effects of early AdVEGF expression may have led to enhanced alloimmune activation and thereby an increase in airway wall expression of endogenous VEGF. Epithelial cells express VEGFR-1 and VEGFR-2, and it is possible that VEGF directly either promotes epithelial regeneration or inhibits epithelial cell death (26). In addition to being directly proproliferative for epithelial cells, VEGF may also elicit its effects indirectly via upregulation of other factors that induce epithelial regeneration or by attenuation of ischemic injury through accelerated revascularization of the allograft. In our previous study, we showed that upregulation of inducible NO synthase activity with L-arginine decreased epithelial necrosis (17). However, in this study, inhibition of the NOe pathway by L-NAME did not inhibit the beneficial effect of VEGF gene transfer in epithelial regeneration, suggesting that the effect of VEGF is not NO-dependent.

The main finding of this study is that VEGF enhances the development of OB. VEGF gene transfer significantly increased tracheal allograft luminal occlusion, whereas inhibition of VEGFR tyrosine kinase activity with PTK787 decreased the development of OB and negated the deleterious effect of VEGF gene transfer. The mechanism by which VEGF accelerates the development of OB seems to be twofold; on one hand, VEGF is proinflammatory, and on the other, proproliferative.

VEGF may exert its proinflammatory role in the transplant setting by inducing the expression of several adhesion molecules (intercellular adhesion molecule 1, vascular cell adhesion molecule 1, E-selectin) (27) and proinflammatory cytokines (monocyte chemoattractant protein-1, interleukin 8) (28) or by direct monocyte/macrophage chemotaxis via VEGFR-1 (9). On the other hand, chronic inflammation induces VEGF-mediated microvascular remodeling and thereby migration of inflammatory cells through the endothelial lining into perivascular spaces (29). Our results support the proinflammatory role for VEGF because blocking the VEGF signaling pathway with PTK787 caused a significant reduction in the number of allograft CD4+ and CD8+ T cells 10 days after transplantation. This antiinflammatory effect of PTK787 treatment did not reduce the number of RECA-1+ vessels in the different treatment groups, indicating that the number of intragraft blood vessels was not affected by VEGF blockade. Our finding is in line with a previous study where chronic mouse Mycoplasma pulmonis infection was associated with increased VEGF expression and dilatation and increased permeability of pulmonary blood vessels but not with formation of new blood vessels (5). In our study, the number of remodeling microvascular blood vessels, as revealed by the presence of activated pericytes, was increased by VEGF overexpression and decreased in PTK787-treated animals, suggesting that VEGF induces and supports active microvascular remodeling. Vascular permeability of remodeling vessels is caused by increased luminal surface area, enhanced susceptibility of these vessels to inflammatory stimuli that induce vascular permeability (30), increased formation of endothelial cell gaps (31), or a change in the endothelial cell phenotype to a more permeable type (32). Furthermore, enlargement of arterioles might increase intravascular pressure in the allograft and lead to enhanced vascular leakage.

Lymphangiogenesis is believed to be mediated mainly by binding of VEGF-C and VEGF-D to their high-affinity receptor, VEGFR-3 (33). A recent study demonstrates that VEGF stimulates lymphangiogenesis and hemangiogenesis in inflammatory neovascularization via recruitment of macrophages (34). Lymphangiogenesis may enhance alloimmune sensitization by increased antigen presentation to the host immune system. In the transplantation setting, an increased lymphatic network could lead to continuous low-grade export of donor tissue to sentinel lymph nodes, resulting in persistant rejection and allograft injury. This study demonstrates that VEGF gene transfer doubled the number of LYVE-1+ lymphatic vessels in the allograft airway wall. On the other hand, VEGFR protein tyrosine kinase inhibitor PTK787 halved the number of LYVE-1+ lymphatic vessels and the number of graft-infiltrating CD4+ and CD8+ T cells in the airway wall. These observations suggest that lymphangiogenesis leads to a sustained alloimmune response and may play a role in the development of OB in this model.

VEGF is not a direct mitogen for SMCs per se but may elicit its proproliferative effects indirectly. VEGF may affect SMC migration and could thereby play a role in the development of OB. Airway SMCs are known to express VEGFR and can produce fibronectin after VEGF stimulation (35). Furthermore, VEGF may increase SMC migration and proliferation indirectly by triggering the release of PDGF from adjacent cells (16, 36–39). We have previously shown that inhibition of PDGFR tyrosine kinase activity reduces OB formation effectively without an antiinflammatory effect (19). In the present study, PDGFR tyrosine kinase inhibition with imatinib totally prevented luminal occlusion in tracheal allografts with AdVEGF gene transfer, suggesting that VEGF activates the PDGF signaling pathway, leading to increased development of OB. In addition, VEGF may induce recruitment of circulating hematopoietic stem cells into the allograft where these cells may differentiate and proliferate into myofibroblasts in the presence of other growth factors, such as transforming growth factor ß and PDGF (40, 41). In support of this, lesional cells seen in bleomycin-mediated pulmonary fibrosis and transplant arteriosclerosis (a manifestation of chronic rejection in cardiac allografts) may also be derived from bone marrow progenitor cells (40, 42, 43).

In conclusion, we show that VEGF expression is upregulated in graft-infiltrating mononuclear inflammatory cells during the development of OB. After transplantation, VEGF has both beneficial and deleterious properties. Although VEGF induces regeneration of allograft epithelium cells from ischemic and alloimmune injury, it increases the development of tracheal luminal occlusion. VEGF increases OB by promoting the alloimmune response, possibly through lymphatic vessel in-growth, and by inducing SMC migration and proliferation through increased PDGF signaling. The results of this study suggest a central and biologically significant role for VEGF in the development of OB and that specific modulation of the VEGF and PDGF signaling pathways may have clinical implications in the future.


   Acknowledgments
 
The authors thank E. Rouvinen, R.N., and E. Wasenius, R.N., for their excellent technical assistance.


   FOOTNOTES
 
Supported by grants from Helsinki University Central Hospital research funds, the Sigrid Juselius Foundation, Finnish Life and Pension Insurance Companies, the Academy of Finland (Project No. 1201292), Finska Läkaresällskapet, Jalmari and Rauha Ahokas Foundation, and the Research and Science Foundation of Farmos.

This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

Conflict of Interest Statement: R.K. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; J.M.T. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; A.I.N. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; J.W. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; M.J. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; S.Y.-H. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; P.K.K. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; K.B.L. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form August 3, 2004; accepted in final form March 15, 2005


   REFERENCES
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 RESULTS
 DISCUSSION
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