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29: Role of Vascular Endothelial Growth Factor-C in Experimental Obliterative Airway Disease
S70
Abstracts
Purpose: Aberrant airway epithelial repair is a key event in the aetiology of obliterative bronchiolitis (OB). EMT is a process where cells lose epithelial properties, become invasive and deposit extracellular matrix (ECM). EMT may be important in the aberrant airway repair in OB. We investigated if TGF, which is abundantly present in OB lung, could induce EMT in airway epithelial cells. Furthermore we assessed if an acute inflammatory insult might influence the epithelial cells response to TGF. Methods and Materials: Primary cultures of bronchial epithelial cells were established from the airways of stable lung transplant recipients. Cells were stimulated with TGF1 (10ng/ml) ⫾ TNF␣ (20ng/ml) for 72 hours and compared to controls. Cell phenotype was monitored and changes in expression of epithelial markers (E-cadherin and Cytokeratin-19), and mesenchymal markers (vimentin and fibronectin) assessed by western blotting and confocal microscopy. Deposition of ECM was assessed by TCA precipitation, matrix metalloproteinases (MMP) activity by zymography and cell invasiveness using Matrigel coated Millipore filters. Results: Untreated cells show typical epithelial morphology with strong E-cadherin and cytokeratin expression. TGF treated cells changed phenotype with loss of epithelial markers and increased expression of vimentin and fibronectin. Treated cells secreted collagen 1 and fibronectin, increased MMP9 production, and invaded matrigel filters. Addition of TNF␣ dramatically accentuated both phenotypic and functional features of EMT, vimentin and fibronectin expression and cell invasiness increased 2 fold compared to TGF alone (p⬍0.05, n⫽4). Conclusions: This study provides the first ex-vivo evidence that epithelial cells from the human allograft airway undergo TGF driven EMT and that acute inflammatory insults can dramatically accentuate this phenomenon. These findings suggest the interaction between TGF and acute inflammation may provide a therapeutic target in OB. 28 Maintenance of Canonical I-B Kinase Activity Is Required To Prevent Lung Graft Injury H.J. Huang,1 S. Sugimoto,2 J. Lai,2 N.A. Das,2 M. Okazaki,2 G.A. Patterson,2 D. Kreisel,2 A.E. Gelman,2 1Department of Internal Medicine, Washington University School of Medicine, Saint Louis, MO; 2Department of Surgery, Washington University School of Medicine, Saint Louis, MO Purpose: Lung graft ischemia reperfusion (I/R) injury is associated with increased expression of NF-B dependent genes that control inflammation and cell survival. The Canonical I-B Kinase (IKK) complex is a pivotal regulator of signal transduction pathways that target NF-B activation. These observations have led to the notion that pharmacological inhibition of IKK activity may be a useful strategy to prevent I/R injury. Using a selective IKK peptide inhibitor (NBD) we asked if IKK activity regulates lung graft I/R injury. Methods and Materials: C57BL/63 C57BL/6 orthotopic vascularized left lung transplantation was conducted after 18 hrs of cold preservation. Five minutes before reperfusion 2 g/g body weight (low dose) or 5 g/g body weight (high dose) of NEMO Binding Domain (NBD) peptide or an analogue peptide control (NBD-C) was administered intravenously to recipients. Recipients were sacrificed 6 or 24hrs later and assessed for measures of graft function and inflammation. Results: Compared to NBD-C treated recipients low dose NBD administration reduced intragraft IKK activity by 56.4⫹/-8.6%, improved lung graft function, attenuated intragraft NF-B dependent inflammatory gene expression and blunted intragraft neutrophil accumulation 6 and 24 hrs following reperfusion. Paradoxically, high dose NBD treatment that reduced IKK activity by 94.1⫹/-3.7 % significantly worsened lung graft function and elevated intragraft
The Journal of Heart and Lung Transplantation February 2008
expression of NF-B dependent inflammatory mediators such as IL-1, IL-6, KC, MIP-1, MCP-1 and RANTES as compared to low dose NBD or NBD-C treated recipients. High dose NBD treatment was also coincident with a higher percentage of graft infiltrating neutrophils and a significant increase of apoptotic capillary endothelial and airway epithelial cells when compared to low dose NBD or NBD-C treated recipients. Conclusions: These data highlight the potential complications of suppressing IKK activity to treat lung graft I/R injury as some basal NF-B activity may be necessary to negatively regulate inflammation and promote cell survival. 29 Role of Vascular Endothelial Growth Factor-C in Experimental Obliterative Airway Disease ¨ nen,1 K. Alitalo,2 R. Krebs,1 J.M. Tikkanen,1 A.I. Nyka 3 1 S. Yla ¨ -Herttuala, P.K. Koskinen, K.B. Lemstro ¨ m,1 1 Transplantation Laboratory, University of Helsinki, Helsinki, Finland; 2Molecular Cancer Biology Laboratory, University of Helsinki, Helsinki, Finland; 3A.I.Virtanen Institute for Molecular Sciences, University of Kuopio, Kuopio, Finland Purpose: The role of vascular endothelial growth factor-C (VEGF-C) in the development of obliterative airway disease (OAD) as a manifestation of chronic rejection was investigated in the rat tracheal allograft model. Methods and Materials: Tracheal allografts were transplanted heterotopically from DA- to WF-rats (syngeneic controls: DA3 DA). Expression of VEGF-C and VEGFR-3 protein and lymphatic vessel marker LYVE-1 was analysed from normal, syngeneic, and allogeneic tracheal transplants. Adenovirally mediated human VEGF-C (AdVEGF-C) gene transfer was used to overexpress VEGF-C (controls: AdLacZ). Cyclosporine A (CyA) was used for background immunosuppression. Tracheal allografts were removed 10 and 30 days after transplantation. Results: An increase in VEGF-C-positive inflammatory cells was observed at 10d (P⬍0.005; n⫽3⫹5) and 30d (P⬍0.01; n⫽5⫹5) allografts when compared to syngeneic grafts. Allografts showed a significantly elevated number of LYVE-1-positive lymphatic vessels at 30d (P⬍0.05; n⫽6⫹6). Adenovirus-mediated human VEGF-C gene transfer into heterotopic tracheal allografts resulted in a more than three-fold increase in airway occlusion at 30d compared to allografts treated with virus expressing -gal (-gal: 17,3 %; VEGF-C: 57,7 %; P⬍0.01; n⫽7⫹9). No significant differences could be found in the numbers of CD4⫹ and CD8⫹ T cells or ED1⫹ macrophages at 10d, the number of myeloperoxidase-producing neutrophils, however, was significantly enhanced in the AdVEGF-C group (P⬍0.05; n⫽8⫹5). Additionally, significant increases of LYVE-1-positive cells (P⬍0.05; n⫽7⫹9) and of CD8positive cells (P⬍0.05; n⫽6⫹9) were observed in AdVEGF-C-treated allografts when compared to AdLacZ-treated allografts at 30d. Conclusions: This study suggests that VEGF-C enhances the posttransplantory inflammatory response possibly via induction of early neutrophilia and enhanced lymphangiogenesis, thereby augmenting the development of OAD. Appropriate inhibition of the VEGF-C signalling pathway may provide a therapeutic strategy for prevention of bronchiolitis obliterans syndrome. 30 CCR2 Is Required for Monocyte Recruitment and Differentiation but Not Acute Rejection of Vascularized Mouse Lung Allografts M. Okazaki,1 S. Sugimoto,1 J. Lai,1 C.G. Kornfeld,1 J.R. Tietjens,1 S.B. Richardson,1 H.J. Huang,1 A. Patterson,1 A.S. Krupnick,1
Abstracts
Purpose: Aberrant airway epithelial repair is a key event in the aetiology of obliterative bronchiolitis (OB). EMT is a process where cells lose epithelial properties, become invasive and deposit extracellular matrix (ECM). EMT may be important in the aberrant airway repair in OB. We investigated if TGF, which is abundantly present in OB lung, could induce EMT in airway epithelial cells. Furthermore we assessed if an acute inflammatory insult might influence the epithelial cells response to TGF. Methods and Materials: Primary cultures of bronchial epithelial cells were established from the airways of stable lung transplant recipients. Cells were stimulated with TGF1 (10ng/ml) ⫾ TNF␣ (20ng/ml) for 72 hours and compared to controls. Cell phenotype was monitored and changes in expression of epithelial markers (E-cadherin and Cytokeratin-19), and mesenchymal markers (vimentin and fibronectin) assessed by western blotting and confocal microscopy. Deposition of ECM was assessed by TCA precipitation, matrix metalloproteinases (MMP) activity by zymography and cell invasiveness using Matrigel coated Millipore filters. Results: Untreated cells show typical epithelial morphology with strong E-cadherin and cytokeratin expression. TGF treated cells changed phenotype with loss of epithelial markers and increased expression of vimentin and fibronectin. Treated cells secreted collagen 1 and fibronectin, increased MMP9 production, and invaded matrigel filters. Addition of TNF␣ dramatically accentuated both phenotypic and functional features of EMT, vimentin and fibronectin expression and cell invasiness increased 2 fold compared to TGF alone (p⬍0.05, n⫽4). Conclusions: This study provides the first ex-vivo evidence that epithelial cells from the human allograft airway undergo TGF driven EMT and that acute inflammatory insults can dramatically accentuate this phenomenon. These findings suggest the interaction between TGF and acute inflammation may provide a therapeutic target in OB. 28 Maintenance of Canonical I-B Kinase Activity Is Required To Prevent Lung Graft Injury H.J. Huang,1 S. Sugimoto,2 J. Lai,2 N.A. Das,2 M. Okazaki,2 G.A. Patterson,2 D. Kreisel,2 A.E. Gelman,2 1Department of Internal Medicine, Washington University School of Medicine, Saint Louis, MO; 2Department of Surgery, Washington University School of Medicine, Saint Louis, MO Purpose: Lung graft ischemia reperfusion (I/R) injury is associated with increased expression of NF-B dependent genes that control inflammation and cell survival. The Canonical I-B Kinase (IKK) complex is a pivotal regulator of signal transduction pathways that target NF-B activation. These observations have led to the notion that pharmacological inhibition of IKK activity may be a useful strategy to prevent I/R injury. Using a selective IKK peptide inhibitor (NBD) we asked if IKK activity regulates lung graft I/R injury. Methods and Materials: C57BL/63 C57BL/6 orthotopic vascularized left lung transplantation was conducted after 18 hrs of cold preservation. Five minutes before reperfusion 2 g/g body weight (low dose) or 5 g/g body weight (high dose) of NEMO Binding Domain (NBD) peptide or an analogue peptide control (NBD-C) was administered intravenously to recipients. Recipients were sacrificed 6 or 24hrs later and assessed for measures of graft function and inflammation. Results: Compared to NBD-C treated recipients low dose NBD administration reduced intragraft IKK activity by 56.4⫹/-8.6%, improved lung graft function, attenuated intragraft NF-B dependent inflammatory gene expression and blunted intragraft neutrophil accumulation 6 and 24 hrs following reperfusion. Paradoxically, high dose NBD treatment that reduced IKK activity by 94.1⫹/-3.7 % significantly worsened lung graft function and elevated intragraft
The Journal of Heart and Lung Transplantation February 2008
expression of NF-B dependent inflammatory mediators such as IL-1, IL-6, KC, MIP-1, MCP-1 and RANTES as compared to low dose NBD or NBD-C treated recipients. High dose NBD treatment was also coincident with a higher percentage of graft infiltrating neutrophils and a significant increase of apoptotic capillary endothelial and airway epithelial cells when compared to low dose NBD or NBD-C treated recipients. Conclusions: These data highlight the potential complications of suppressing IKK activity to treat lung graft I/R injury as some basal NF-B activity may be necessary to negatively regulate inflammation and promote cell survival. 29 Role of Vascular Endothelial Growth Factor-C in Experimental Obliterative Airway Disease ¨ nen,1 K. Alitalo,2 R. Krebs,1 J.M. Tikkanen,1 A.I. Nyka 3 1 S. Yla ¨ -Herttuala, P.K. Koskinen, K.B. Lemstro ¨ m,1 1 Transplantation Laboratory, University of Helsinki, Helsinki, Finland; 2Molecular Cancer Biology Laboratory, University of Helsinki, Helsinki, Finland; 3A.I.Virtanen Institute for Molecular Sciences, University of Kuopio, Kuopio, Finland Purpose: The role of vascular endothelial growth factor-C (VEGF-C) in the development of obliterative airway disease (OAD) as a manifestation of chronic rejection was investigated in the rat tracheal allograft model. Methods and Materials: Tracheal allografts were transplanted heterotopically from DA- to WF-rats (syngeneic controls: DA3 DA). Expression of VEGF-C and VEGFR-3 protein and lymphatic vessel marker LYVE-1 was analysed from normal, syngeneic, and allogeneic tracheal transplants. Adenovirally mediated human VEGF-C (AdVEGF-C) gene transfer was used to overexpress VEGF-C (controls: AdLacZ). Cyclosporine A (CyA) was used for background immunosuppression. Tracheal allografts were removed 10 and 30 days after transplantation. Results: An increase in VEGF-C-positive inflammatory cells was observed at 10d (P⬍0.005; n⫽3⫹5) and 30d (P⬍0.01; n⫽5⫹5) allografts when compared to syngeneic grafts. Allografts showed a significantly elevated number of LYVE-1-positive lymphatic vessels at 30d (P⬍0.05; n⫽6⫹6). Adenovirus-mediated human VEGF-C gene transfer into heterotopic tracheal allografts resulted in a more than three-fold increase in airway occlusion at 30d compared to allografts treated with virus expressing -gal (-gal: 17,3 %; VEGF-C: 57,7 %; P⬍0.01; n⫽7⫹9). No significant differences could be found in the numbers of CD4⫹ and CD8⫹ T cells or ED1⫹ macrophages at 10d, the number of myeloperoxidase-producing neutrophils, however, was significantly enhanced in the AdVEGF-C group (P⬍0.05; n⫽8⫹5). Additionally, significant increases of LYVE-1-positive cells (P⬍0.05; n⫽7⫹9) and of CD8positive cells (P⬍0.05; n⫽6⫹9) were observed in AdVEGF-C-treated allografts when compared to AdLacZ-treated allografts at 30d. Conclusions: This study suggests that VEGF-C enhances the posttransplantory inflammatory response possibly via induction of early neutrophilia and enhanced lymphangiogenesis, thereby augmenting the development of OAD. Appropriate inhibition of the VEGF-C signalling pathway may provide a therapeutic strategy for prevention of bronchiolitis obliterans syndrome. 30 CCR2 Is Required for Monocyte Recruitment and Differentiation but Not Acute Rejection of Vascularized Mouse Lung Allografts M. Okazaki,1 S. Sugimoto,1 J. Lai,1 C.G. Kornfeld,1 J.R. Tietjens,1 S.B. Richardson,1 H.J. Huang,1 A. Patterson,1 A.S. Krupnick,1