Dual Role of Vascular Endothelial Growth Factor in Experimental Obliterative Bronchiolitis
http://www.100md.com
美国呼吸和危急护理医学 2005年第6期
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
Department of Cardiothoracic Surgery, Helsinki University Central Hospital, Helsinki
A.I. Virtanen Institute for Molecular Sciences, University of Kuopio, Kuopio, Finland
Novartis Pharma, Basel, Switzerland
ABSTRACT
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 celleCspecific 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
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 -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 -galactosidaseeCencoding adenovirus for whole-mount X-gal staining and with enhanced green fluorescent protein (EGFP)eCencoding 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 antieChigh-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 antieCLYVE-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
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).
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.
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 -galactosidase and EGFP-expression (Figures 3B, 3D, and 3F). At 7 days, the level of infection was already reduced although mild epithelial -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 -galactosidase activity nor EGFP fluorescent signal was observed from the liver or the surrounding omentum at any time point (data not shown).
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).
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).
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 antigeneCpositive immunostaining for activated pericytes (p < 0.05) and the number of LYVE-1+ lymphatic vessels, when compared with appropriate controls, respectively (Figure 7).
DISCUSSION
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 -SMC actineCpositive 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, 36eC39). 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 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.
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
REFERENCES
Hertz MI, Taylor DO, Trulock EP, Boucek MM, Mohacsi PJ, Edwards LB, Keck BM. The registry of the International Society for Heart and Lung Transplantation: nineteenth official report—2002. J Heart Lung Transplant 2002;21:950eC970.
Yousem SA, Berry GJ, Cagle PT, Chamberlain D, Husain AN, Hruban RH, Marchevsky A, Ohori NP, Ritter J, Stewart S, et al. Revision of the 1990 working formulation for the classification of pulmonary allograft rejection: Lung Rejection Study Group. J Heart Lung Transplant 1996;15:1eC15.
Trulock EP. Lung transplantation. Am J Respir Crit Care Med 1997;155:789eC818.
Hoshino M, Takahashi M, Aoike N. Expression of vascular endothelial growth factor, basic fibroblast growth factor, and angiogenin immunoreactivity in asthmatic airways and its relationship to angiogenesis. J Allergy Clin Immunol 2001;107:295eC301.
Thurston G, Murphy TJ, Baluk P, Lindsey JR, McDonald DM. Angiogenesis in mice with chronic airway inflammation: strain-dependent differences. Am J Pathol 1998;153:1099eC1112.
Ferrara N. Molecular and biological properties of vascular endothelial growth factor. J Mol Med 1999;77:527eC543.
Detmar M, Brown LF, Schon MP, Elicker BM, Velasco P, Richard L, Fukumura D, Monsky W, Claffey KP, Jain RK. Increased microvascular density and enhanced leukocyte rolling and adhesion in the skin of VEGF transgenic mice. J Invest Dermatol 1998;111:1eC6.
Melder RJ, Koenig GC, Witwer BP, Safabakhsh N, Munn LL, Jain RK. During angiogenesis, vascular endothelial growth factor and basic fibroblast growth factor regulate natural killer cell adhesion to tumor endothelium. Nat Med 1996;2:992eC997.
Barleon B, Sozzani S, Zhou D, Weich HA, Mantovani A, Marme D. Migration of human monocytes in response to vascular endothelial growth factor (VEGF) is mediated via the VEGF receptor flt-1. Blood 1996;87:3336eC3343.
Brown KR, England KM, Goss KL, Snyder JM, Acarregui MJ. VEGF induces airway epithelial cell proliferation in human fetal lung in vitro. Am J Physiol Lung Cell Mol Physiol 2001;281:L1001eCL1010.
Lee CG, Link H, Baluk P, Homer RJ, Chapoval S, Bhandari V, Kang MJ, Cohn L, Kim YK, McDonald DM, et al. Vascular endothelial growth factor (VEGF) induces remodeling and enhances TH2-mediated sensitization and inflammation in the lung. Nat Med 2004;10:1095eC1103.
Tikkanen JM, Kallio EA, Bruggeman CA, Koskinen PK, Lemstrom KB. Prevention of cytomegalovirus infection-enhanced experimental obliterative bronchiolitis by antiviral prophylaxis or immunosuppression in rat tracheal allografts. Am J Respir Crit Care Med 2001;164:672eC679.
Hiltunen MO, Turunen MP, Turunen AM, Rissanen TT, Laitinen M, Kosma VM, Yla-Herttuala S. Biodistribution of adenoviral vector to nontarget tissues after local in vivo gene transfer to arterial wall using intravascular and periadventitial gene delivery methods. FASEB J 2000;14:2230eC2236.
Silvestre JS, Tamarat R, Ebrahimian TG, Le-Roux A, Clergue M, Emmanuel F, Duriez M, Schwartz B, Branellec D, Levy BI. Vascular endothelial growth factor-B promotes in vivo angiogenesis. Circ Res 2003;93:114eC123.
Schlingemann RO, Rietveld FJ, de Waal RM, Ferrone S, Ruiter DJ. Expression of the high molecular weight melanoma-associated antigen by pericytes during angiogenesis in tumors and in healing wounds. Am J Pathol 1990;136:1393eC1405.
Lemstrom KB, Krebs R, Nykanen AI, Tikkanen JM, Sihvola RK, Aaltola EM, Hayry PJ, Wood J, Alitalo K, Yla-Herttuala S, et al. Vascular endothelial growth factor enhances cardiac allograft arteriosclerosis. Circulation 2002;105:2524eC2530.
Kallio EA, Koskinen PK, Aavik E, Vaali K, Lemstom KB. Role of nitric oxide in experimental obliterative bronchiolitis (chronic rejection) in the rat. J Clin Invest 1997;100:2984eC2994.
van der Zee R, Murohara T, Luo Z, Zollmann F, Passeri J, Lekutat C, Isner JM. Vascular endothelial growth factor/vascular permeability factor augments nitric oxide release from quiescent rabbit and human vascular endothelium. Circulation 1997;95:1030eC1037.
Kallio EA, Koskinen PK, Aavik E, Buchdunger E, Lemstrom KB. Role of platelet-derived growth factor in obliterative bronchiolitis (chronic rejection) in the rat. Am J Respir Crit Care Med 1999;160:1324eC1332.
Hele DJ, Yacoub MH, Belvisi MG. The heterotopic tracheal allograft as an animal model of obliterative bronchiolitis. Respir Res 2001;2:169eC183.
Koskinen PK, Kallio EA, Krebs R, Lemstrom KB. A dose-dependent inhibitory effect of cyclosporine A on obliterative bronchiolitis of rat tracheal allografts. Am J Respir Crit Care Med 1997;155:303eC312.
Fehrenbach A, Pufe T, Wittwer T, Nagib R, Dreyer N, Pech T, Petersen W, Fehrenbach H, Wahlers T, Richter J. Reduced vascular endothelial growth factor correlates with alveolar epithelial damage after experimental ischemia and reperfusion. J Heart Lung Transplant 2003;22:967eC978.
Meyer KC, Cardoni AL, Xiang Z, Cornwell RD, Love RB. Vascular endothelial growth factor in human lung transplantation. Chest 2001;119:137eC143.
Reinders M, Fang JC, Wong W, Ganz P, Briscoe DM. Expression patterns of vascular endothelial growth factor in human cardiac allografts: association with rejection. Transplantation 2003;76:224eC230.
Ozawa CR, Banfi A, Glazer NL, Thurston G, Springer ML, Kraft PE, McDonald DM, Blau HM. Microenvironmental VEGF concentration, not total dose, determines a threshold between normal and aberrant angiogenesis. J Clin Invest 2004;113:516eC527.
Lassus P, Turanlahti M, Heikkila P, Andersson LC, Nupponen I, Sarnesto A, Andersson S. Pulmonary vascular endothelial growth factor and Flt-1 in fetuses, in acute and chronic lung disease, and in persistent pulmonary hypertension of the newborn. Am J Respir Crit Care Med 2001;164:1981eC1987.
Kim I, Moon SO, Kim SH, Kim HJ, Koh YS, Koh GY. Vascular endothelial growth factor expression of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and E-selectin through nuclear factor-kappa B activation in endothelial cells. J Biol Chem 2001;276:7614eC7620.
Reinders ME, Sho M, Izawa A, Wang P, Mukhopadhyay D, Koss KE, Geehan CS, Luster AD, Sayegh MH, Briscoe DM. Proinflammatory functions of vascular endothelial growth factor in alloimmunity. J Clin Invest 2003;112:1655eC1665.
McDonald DM. Angiogenesis and remodeling of airway vasculature in chronic inflammation. Am J Respir Crit Care Med 2001;164:S39eCS45.
Kwan ML, Gomez AD, Baluk P, Hashizume H, McDonald DM. Airway vasculature after mycoplasma infection: chronic leakiness and selective hypersensitivity to substance P. Am J Physiol Lung Cell Mol Physiol 2001;280:L286eCL297.
McDonald DM. Endothelial gaps and permeability of venules in rat tracheas exposed to inflammatory stimuli. Am J Physiol 1994;266:L61eCL83.
Baluk P, Bowden JJ, Lefevre PM, McDonald DM. Upregulation of substance P receptors in angiogenesis associated with chronic airway inflammation in rats. Am J Physiol 1997;273:L565eCL571.
Veikkola T, Jussila L, Makinen T, Karpanen T, Jeltsch M, Petrova TV, Kubo H, Thurston G, McDonald DM, Achen MG, et al. Signalling via vascular endothelial growth factor receptor-3 is sufficient for lymphangiogenesis in transgenic mice. EMBO J 2001;20:1223eC1231.
Cursiefen C, Chen L, Borges LP, Jackson D, Cao J, Radziejewski C, D'Amore PA, Dana MR, Wiegand SJ, Streilein JW. VEGF-A stimulates lymphangiogenesis and hemangiogenesis in inflammatory neovascularization via macrophage recruitment. J Clin Invest 2004;113:1040eC1050.
Kazi AS, Lotfi S, Goncharova EA, Tliba O, Amrani Y, Krymskaya VP, Lazaar AL. Vascular endothelial growth factor-induced secretion of fibronectin is ERK dependent. Am J Physiol Lung Cell Mol Physiol 2004;286:L539eCL545.
Cucina A, Borrelli V, Randone B, Coluccia P, Sapienza P, Cavallaro A. Vascular endothelial growth factor increases the migration and proliferation of smooth muscle cells through the mediation of growth factors released by endothelial cells. J Surg Res 2003;109:16eC23.
Grosskreutz CL, Anand-Apte B, Duplaa C, Quinn TP, Terman BI, Zetter B, D'Amore PA. Vascular endothelial growth factor-induced migration of vascular smooth muscle cells in vitro. Microvasc Res 1999;58:128eC136.
Hirschi KK, Rohovsky SA, D'Amore PA. PDGF, TGF-beta, and heterotypic cell-cell interactions mediate endothelial cell-induced recruitment of 10T1/2 cells and their differentiation to a smooth muscle fate. J Cell Biol 1998;141:805eC814.
Wang H, Keiser JA. Vascular endothelial growth factor upregulates the expression of matrix metalloproteinases in vascular smooth muscle cells: role of flt-1. Circ Res 1998;83:832eC840.
Hashimoto N, Jin H, Liu T, Chensue SW, Phan SH. Bone marrow-derived progenitor cells in pulmonary fibrosis. J Clin Invest 2004;113:243eC252.
Hattori K, Dias S, Heissig B, Hackett NR, Lyden D, Tateno M, Hicklin DJ, Zhu Z, Witte L, Crystal RG, et al. Vascular endothelial growth factor and angiopoietin-1 stimulate postnatal hematopoiesis by recruitment of vasculogenic and hematopoietic stem cells. J Exp Med 2001;193:1005eC1014.
Epperly MW, Guo H, Gretton JE, Greenberger JS. Bone marrow origin of myofibroblasts in irradiation pulmonary fibrosis. Am J Respir Cell Mol Biol 2003;29:213eC224.
Hillebrands J-L, Klatter FA, Rozing J. Origin of vascular smooth muscle cells and the role of circulating stem cells in transplant arteriosclerosis. Arterioscler Thromb Vasc Biol 2003;23:380eC387.(Rainer Krebs, Jussi M. Ti)
Molecular Cancer Biology Laboratory, Biomedicum Helsinki, University of Helsinki
Division of Nephrology, Department of Medicine
Department of Cardiothoracic Surgery, Helsinki University Central Hospital, Helsinki
A.I. Virtanen Institute for Molecular Sciences, University of Kuopio, Kuopio, Finland
Novartis Pharma, Basel, Switzerland
ABSTRACT
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 celleCspecific 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
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 -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 -galactosidaseeCencoding adenovirus for whole-mount X-gal staining and with enhanced green fluorescent protein (EGFP)eCencoding 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 antieChigh-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 antieCLYVE-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
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).
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.
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 -galactosidase and EGFP-expression (Figures 3B, 3D, and 3F). At 7 days, the level of infection was already reduced although mild epithelial -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 -galactosidase activity nor EGFP fluorescent signal was observed from the liver or the surrounding omentum at any time point (data not shown).
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).
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).
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 antigeneCpositive immunostaining for activated pericytes (p < 0.05) and the number of LYVE-1+ lymphatic vessels, when compared with appropriate controls, respectively (Figure 7).
DISCUSSION
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 -SMC actineCpositive 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, 36eC39). 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 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.
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
REFERENCES
Hertz MI, Taylor DO, Trulock EP, Boucek MM, Mohacsi PJ, Edwards LB, Keck BM. The registry of the International Society for Heart and Lung Transplantation: nineteenth official report—2002. J Heart Lung Transplant 2002;21:950eC970.
Yousem SA, Berry GJ, Cagle PT, Chamberlain D, Husain AN, Hruban RH, Marchevsky A, Ohori NP, Ritter J, Stewart S, et al. Revision of the 1990 working formulation for the classification of pulmonary allograft rejection: Lung Rejection Study Group. J Heart Lung Transplant 1996;15:1eC15.
Trulock EP. Lung transplantation. Am J Respir Crit Care Med 1997;155:789eC818.
Hoshino M, Takahashi M, Aoike N. Expression of vascular endothelial growth factor, basic fibroblast growth factor, and angiogenin immunoreactivity in asthmatic airways and its relationship to angiogenesis. J Allergy Clin Immunol 2001;107:295eC301.
Thurston G, Murphy TJ, Baluk P, Lindsey JR, McDonald DM. Angiogenesis in mice with chronic airway inflammation: strain-dependent differences. Am J Pathol 1998;153:1099eC1112.
Ferrara N. Molecular and biological properties of vascular endothelial growth factor. J Mol Med 1999;77:527eC543.
Detmar M, Brown LF, Schon MP, Elicker BM, Velasco P, Richard L, Fukumura D, Monsky W, Claffey KP, Jain RK. Increased microvascular density and enhanced leukocyte rolling and adhesion in the skin of VEGF transgenic mice. J Invest Dermatol 1998;111:1eC6.
Melder RJ, Koenig GC, Witwer BP, Safabakhsh N, Munn LL, Jain RK. During angiogenesis, vascular endothelial growth factor and basic fibroblast growth factor regulate natural killer cell adhesion to tumor endothelium. Nat Med 1996;2:992eC997.
Barleon B, Sozzani S, Zhou D, Weich HA, Mantovani A, Marme D. Migration of human monocytes in response to vascular endothelial growth factor (VEGF) is mediated via the VEGF receptor flt-1. Blood 1996;87:3336eC3343.
Brown KR, England KM, Goss KL, Snyder JM, Acarregui MJ. VEGF induces airway epithelial cell proliferation in human fetal lung in vitro. Am J Physiol Lung Cell Mol Physiol 2001;281:L1001eCL1010.
Lee CG, Link H, Baluk P, Homer RJ, Chapoval S, Bhandari V, Kang MJ, Cohn L, Kim YK, McDonald DM, et al. Vascular endothelial growth factor (VEGF) induces remodeling and enhances TH2-mediated sensitization and inflammation in the lung. Nat Med 2004;10:1095eC1103.
Tikkanen JM, Kallio EA, Bruggeman CA, Koskinen PK, Lemstrom KB. Prevention of cytomegalovirus infection-enhanced experimental obliterative bronchiolitis by antiviral prophylaxis or immunosuppression in rat tracheal allografts. Am J Respir Crit Care Med 2001;164:672eC679.
Hiltunen MO, Turunen MP, Turunen AM, Rissanen TT, Laitinen M, Kosma VM, Yla-Herttuala S. Biodistribution of adenoviral vector to nontarget tissues after local in vivo gene transfer to arterial wall using intravascular and periadventitial gene delivery methods. FASEB J 2000;14:2230eC2236.
Silvestre JS, Tamarat R, Ebrahimian TG, Le-Roux A, Clergue M, Emmanuel F, Duriez M, Schwartz B, Branellec D, Levy BI. Vascular endothelial growth factor-B promotes in vivo angiogenesis. Circ Res 2003;93:114eC123.
Schlingemann RO, Rietveld FJ, de Waal RM, Ferrone S, Ruiter DJ. Expression of the high molecular weight melanoma-associated antigen by pericytes during angiogenesis in tumors and in healing wounds. Am J Pathol 1990;136:1393eC1405.
Lemstrom KB, Krebs R, Nykanen AI, Tikkanen JM, Sihvola RK, Aaltola EM, Hayry PJ, Wood J, Alitalo K, Yla-Herttuala S, et al. Vascular endothelial growth factor enhances cardiac allograft arteriosclerosis. Circulation 2002;105:2524eC2530.
Kallio EA, Koskinen PK, Aavik E, Vaali K, Lemstom KB. Role of nitric oxide in experimental obliterative bronchiolitis (chronic rejection) in the rat. J Clin Invest 1997;100:2984eC2994.
van der Zee R, Murohara T, Luo Z, Zollmann F, Passeri J, Lekutat C, Isner JM. Vascular endothelial growth factor/vascular permeability factor augments nitric oxide release from quiescent rabbit and human vascular endothelium. Circulation 1997;95:1030eC1037.
Kallio EA, Koskinen PK, Aavik E, Buchdunger E, Lemstrom KB. Role of platelet-derived growth factor in obliterative bronchiolitis (chronic rejection) in the rat. Am J Respir Crit Care Med 1999;160:1324eC1332.
Hele DJ, Yacoub MH, Belvisi MG. The heterotopic tracheal allograft as an animal model of obliterative bronchiolitis. Respir Res 2001;2:169eC183.
Koskinen PK, Kallio EA, Krebs R, Lemstrom KB. A dose-dependent inhibitory effect of cyclosporine A on obliterative bronchiolitis of rat tracheal allografts. Am J Respir Crit Care Med 1997;155:303eC312.
Fehrenbach A, Pufe T, Wittwer T, Nagib R, Dreyer N, Pech T, Petersen W, Fehrenbach H, Wahlers T, Richter J. Reduced vascular endothelial growth factor correlates with alveolar epithelial damage after experimental ischemia and reperfusion. J Heart Lung Transplant 2003;22:967eC978.
Meyer KC, Cardoni AL, Xiang Z, Cornwell RD, Love RB. Vascular endothelial growth factor in human lung transplantation. Chest 2001;119:137eC143.
Reinders M, Fang JC, Wong W, Ganz P, Briscoe DM. Expression patterns of vascular endothelial growth factor in human cardiac allografts: association with rejection. Transplantation 2003;76:224eC230.
Ozawa CR, Banfi A, Glazer NL, Thurston G, Springer ML, Kraft PE, McDonald DM, Blau HM. Microenvironmental VEGF concentration, not total dose, determines a threshold between normal and aberrant angiogenesis. J Clin Invest 2004;113:516eC527.
Lassus P, Turanlahti M, Heikkila P, Andersson LC, Nupponen I, Sarnesto A, Andersson S. Pulmonary vascular endothelial growth factor and Flt-1 in fetuses, in acute and chronic lung disease, and in persistent pulmonary hypertension of the newborn. Am J Respir Crit Care Med 2001;164:1981eC1987.
Kim I, Moon SO, Kim SH, Kim HJ, Koh YS, Koh GY. Vascular endothelial growth factor expression of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and E-selectin through nuclear factor-kappa B activation in endothelial cells. J Biol Chem 2001;276:7614eC7620.
Reinders ME, Sho M, Izawa A, Wang P, Mukhopadhyay D, Koss KE, Geehan CS, Luster AD, Sayegh MH, Briscoe DM. Proinflammatory functions of vascular endothelial growth factor in alloimmunity. J Clin Invest 2003;112:1655eC1665.
McDonald DM. Angiogenesis and remodeling of airway vasculature in chronic inflammation. Am J Respir Crit Care Med 2001;164:S39eCS45.
Kwan ML, Gomez AD, Baluk P, Hashizume H, McDonald DM. Airway vasculature after mycoplasma infection: chronic leakiness and selective hypersensitivity to substance P. Am J Physiol Lung Cell Mol Physiol 2001;280:L286eCL297.
McDonald DM. Endothelial gaps and permeability of venules in rat tracheas exposed to inflammatory stimuli. Am J Physiol 1994;266:L61eCL83.
Baluk P, Bowden JJ, Lefevre PM, McDonald DM. Upregulation of substance P receptors in angiogenesis associated with chronic airway inflammation in rats. Am J Physiol 1997;273:L565eCL571.
Veikkola T, Jussila L, Makinen T, Karpanen T, Jeltsch M, Petrova TV, Kubo H, Thurston G, McDonald DM, Achen MG, et al. Signalling via vascular endothelial growth factor receptor-3 is sufficient for lymphangiogenesis in transgenic mice. EMBO J 2001;20:1223eC1231.
Cursiefen C, Chen L, Borges LP, Jackson D, Cao J, Radziejewski C, D'Amore PA, Dana MR, Wiegand SJ, Streilein JW. VEGF-A stimulates lymphangiogenesis and hemangiogenesis in inflammatory neovascularization via macrophage recruitment. J Clin Invest 2004;113:1040eC1050.
Kazi AS, Lotfi S, Goncharova EA, Tliba O, Amrani Y, Krymskaya VP, Lazaar AL. Vascular endothelial growth factor-induced secretion of fibronectin is ERK dependent. Am J Physiol Lung Cell Mol Physiol 2004;286:L539eCL545.
Cucina A, Borrelli V, Randone B, Coluccia P, Sapienza P, Cavallaro A. Vascular endothelial growth factor increases the migration and proliferation of smooth muscle cells through the mediation of growth factors released by endothelial cells. J Surg Res 2003;109:16eC23.
Grosskreutz CL, Anand-Apte B, Duplaa C, Quinn TP, Terman BI, Zetter B, D'Amore PA. Vascular endothelial growth factor-induced migration of vascular smooth muscle cells in vitro. Microvasc Res 1999;58:128eC136.
Hirschi KK, Rohovsky SA, D'Amore PA. PDGF, TGF-beta, and heterotypic cell-cell interactions mediate endothelial cell-induced recruitment of 10T1/2 cells and their differentiation to a smooth muscle fate. J Cell Biol 1998;141:805eC814.
Wang H, Keiser JA. Vascular endothelial growth factor upregulates the expression of matrix metalloproteinases in vascular smooth muscle cells: role of flt-1. Circ Res 1998;83:832eC840.
Hashimoto N, Jin H, Liu T, Chensue SW, Phan SH. Bone marrow-derived progenitor cells in pulmonary fibrosis. J Clin Invest 2004;113:243eC252.
Hattori K, Dias S, Heissig B, Hackett NR, Lyden D, Tateno M, Hicklin DJ, Zhu Z, Witte L, Crystal RG, et al. Vascular endothelial growth factor and angiopoietin-1 stimulate postnatal hematopoiesis by recruitment of vasculogenic and hematopoietic stem cells. J Exp Med 2001;193:1005eC1014.
Epperly MW, Guo H, Gretton JE, Greenberger JS. Bone marrow origin of myofibroblasts in irradiation pulmonary fibrosis. Am J Respir Cell Mol Biol 2003;29:213eC224.
Hillebrands J-L, Klatter FA, Rozing J. Origin of vascular smooth muscle cells and the role of circulating stem cells in transplant arteriosclerosis. Arterioscler Thromb Vasc Biol 2003;23:380eC387.(Rainer Krebs, Jussi M. Ti)