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Development of Recipient-Matched Engineered Heart Tissue Using 3D Printing
Abstracts S97 2( 50) Ligation of Transgenic MHC Class I Molecule Expressed Only in the Lungs By Its Specific Antibodies Induces Epithelial Injury, Autoimmunity and Obliterative Airway Disease (OAD): A Novel Transgenic Mouse Model of OAD N.J. Sarma , V. Tiriveedhi, T. Mohanakumar. Surgery, Washington University School of Medicine, St. Louis, MO. Purpose: Antibodies to mismatched Donor HLA induces autoimmunity to lung associated self-antigens Kα 1 Tubulin and Collagen V (SAgs) leading to chronic rejection following lung transplantation (LTx) and BOS. However, the mechanism by which anti-MHC induces epithelial cell damage and BOS is not well understood. Lung specific Clara cells secrete anti-inflammatory Clara cell secretory protein (CCSP) to protect the bronchiolar epithelium. We hypothesize that anti-MHC induced epithelial injury primarily affects the Clara cell leading to OAD in a novel transgenic mouse model in which H2KD is selectively expressed on lung parenchyma. Methods: Transgenic mouse model with lung specific MHC expression was obtained by cloning H2KD under the CCSP promoter. Expression of H2KD was monitored by western blot and qPCR. Anti-MHC class I or control antibodies were administered intravenously. The lungs were harvested on day 15 and 30 for histopathological analysis. Morphometric analysis of Cellular infiltration, epithelial abnormalities and fibrosis was determined by H&E and Trichome staining. The alloimmune responses to the transgenic MHC and the induction of autoimmune responses to SAgs were determined by ELISA. The frequency of SAg specific IFN-γ , IL-10 and IL-17 secreting T cells were enumerated using ELISpot. Results: The transgenic mouse model demonstrated lung specific expression of H2KD. Intravenous administration of anti-H2KD developed marked cellular infiltration around vessels and bronchiole of lung by day 15 leading to epithelial injury, fibrosis, and occlusion of the distal airways by day 30 (OAD) similar to BOS following human LTx. This was accompanied by marked decreased in CCSP expression in the lungs. Transgenic animals receiving the anti-H2KD developed antibodies to SAgs on day 30 with increases in the frequency of SAg specific IFN-γ , IL-4 and IL-17 T cells. Conclusion: Using a novel transgenic mouse model in which foreign MHC is expressed in the lung this study demonstrate that anti-MHC primarily induces damage to Clara cells leading to marked decrease in CCSP resulting in the development of autoimmunity and OAD. This model will be an important tool to understand the role of Clara cells in antibody mediated cellular injury resulting in chronic rejection.
2( 51) Development of Recipient-Matched Engineered Heart Tissue Using 3D Printing Y. Yildirim ,1 S. Pecha,1 H. Naito,2 B. Karikkineth,2 W. Zimmermann,3 H. Reichenspurner,1 T. Eschenhagen.4 1Cardiac Surgery, University Heart Center Hamburg, Hamburg, Germany; 2Department of Experimental Pharmacology and Toxicology, University Medical Center HamburgEppendorf, Hamburg, Germany; 3Pharmacology, University Medical Center Göttingen, Göttingen, Germany; 4Department of Experimental Pharmacology and Toxicology, University Medical Center HAmburgEppendorf, Hamburg, Germany. Purpose: Engineered pouch-like heart muscle constructs can be applied as heart-embracing cardiac grafts in vivo and might be used as biological ventricular assist devices (BioVAD®). Implantation studies showed micro-gaps between the graft and native heart due to a shape mismatch. The incoherence can lead to electrical coupling problems and loss of support force. Here we developed a procedure to create BioVADs individually matched to the recipient heart. Methods: In group A, small animal magnetic resonance imaging (MRI) heart scan was performed in female wistar rats (n= 10). 3D reconstruction of diastolic MRI data was performed and used to create a three-dimensional model of the heart by 3D printing. We used the recipient-matched 3D heart model and grew BioVADs from neonatal rat heart cells, fibrinogen and
serum-containing culture medium. In group B, instead of the 3D heart model, a spheric glas spacer (diameter 10mm) was used to construct pouch like BioVADs. Results: The engineered tissue beat spontaneously and showed contractile properties of native heart muscle including positive inotropic responses to calcium and isoprenaline. In group A recipient heart matched BioVADs were implanted into the corresponding healthy rats (n= 1 0), while in group B the spheric BioVADs were implanted (n= 10). 14 days after implantation hearts were explanted for histology. Histological analysis of recipient-matched BioVADs showed excellent coverage of the epicardial heart surface, while spheric BioVADs showed high number of micro and macro gaps. Furthermore direct alignment of the BioVADs and recipients hearts was measured in cross-sectioned histological compounds. In group B, a statistically higher number of areas with loss of contact between BioVADs and recipient hearts was seen compared to group A. Conclusion: We developed a novel casting technology using small animal MRI and 3D printing to generate artificial cardiac tissue that is perfectly matched to the recipient heart and exhibits structural and functional properties of native myocardium.
2( 52) DAP12 Expression By Lung-Resident Macrophages Mediates Pulmonary Ischemia Reperfusion Injury By Promoting Neutrophil Extravasation J. Spahn ,1 W. Li,1 A.C. Bribriesco,1 J. Liu,1 B. Zinselmeyer,2 H. Shen,3 S.L. Brody,2 D. Goldstein,3 A.S. Krupnick,1 A. Gelman,1 D. Kreisel.1 1Surgery, Washington University St. Louis, St. Louis, MO; 2Medicine, Washington University St. Louis, St. Louis, MO; 3Medicine, Yale University, New Haven, CT. Purpose: Neutrophils infiltrate lung grafts early after reperfusion and are important mediators of primary graft dysfunction. Immune pathways that regulate neutrophil recruitment to lungs during ischemia reperfusion injury remain poorly understood. The purpose of this study was to examine the role of DAP12, an adaptor molecule that regulates innate immune responses, in mediating ischemia reperfusion injury after lung transplantation. Methods: We used a mouse model of orthotopic vascularized lung transplantation. Grafts were assessed functionally (arterial blood gases), immunohistochemically, by quantitative PCR, flow cytometry and intravital two-photon imaging. Chemokine production by macrophages in vitro was assessed by ELISA. Results: We found that macrophages in mouse (qPCR and immunohistochemistry) and human (immunohistochemistry) lungs that have been subjected to cold ischemia express DAP12. Donor, but not recipient deficiency in DAP12 protected against pulmonary ischemia reperfusion injury as evidenced by improved graft function (WT-> WT, mean paO2= 1 80.8 +/-18.9, n= 1 4, DAP12KO-> W T, mean paO2= 410.8 +/-36.9, n= 8, WT-> DAP12KO mean paO2= 217.9 +/-55.9, n= 7 ). Compared to wild-type (WT) B6 cells, DAP12KO bone marrow-derived macrophages produced lower amounts of the neutrophil chemoattractant CXCL2 after in vitro stimulation (17 hours) with lysates derived from syngeneic B6 lung transplants (n= 2 ). Similarly, quantitative PCR of sorted donor macrophages 2 hours after reperfusion in vivo revealed significantly lower expression of CXCL2 when DAP12 was absent (n= 4 ). Intravital two-photon imaging using LysM-GFP neutrophil reporter mice as recipients demonstrated a transendothelial migration defect of neutrophils into DAP12KO lungs at 4 hours after reperfusion (percentage of extravascular neutrophils: WT-> LysM-GFP= 6 9.3%, n= 3 ; DAP12KO-> LysM-GFP= 2 5.6%, n= 3). Neutrophil entry into DAP12-deficient lungs was restored when WT bone-marrow derived macrophages (76.8%, n= 3) or recombinant CXCL2 (63.3%, n= 3) was administered into the donor bronchus prior to reperfusion. Conclusion: Our study has uncovered a previously unrecognized role for DAP12 expression in tissue-resident macrophages in mediating ischemia reperfusion injury after lung transplantation through regulation of neutrophil trafficking.
2( 51) Development of Recipient-Matched Engineered Heart Tissue Using 3D Printing Y. Yildirim ,1 S. Pecha,1 H. Naito,2 B. Karikkineth,2 W. Zimmermann,3 H. Reichenspurner,1 T. Eschenhagen.4 1Cardiac Surgery, University Heart Center Hamburg, Hamburg, Germany; 2Department of Experimental Pharmacology and Toxicology, University Medical Center HamburgEppendorf, Hamburg, Germany; 3Pharmacology, University Medical Center Göttingen, Göttingen, Germany; 4Department of Experimental Pharmacology and Toxicology, University Medical Center HAmburgEppendorf, Hamburg, Germany. Purpose: Engineered pouch-like heart muscle constructs can be applied as heart-embracing cardiac grafts in vivo and might be used as biological ventricular assist devices (BioVAD®). Implantation studies showed micro-gaps between the graft and native heart due to a shape mismatch. The incoherence can lead to electrical coupling problems and loss of support force. Here we developed a procedure to create BioVADs individually matched to the recipient heart. Methods: In group A, small animal magnetic resonance imaging (MRI) heart scan was performed in female wistar rats (n= 10). 3D reconstruction of diastolic MRI data was performed and used to create a three-dimensional model of the heart by 3D printing. We used the recipient-matched 3D heart model and grew BioVADs from neonatal rat heart cells, fibrinogen and
serum-containing culture medium. In group B, instead of the 3D heart model, a spheric glas spacer (diameter 10mm) was used to construct pouch like BioVADs. Results: The engineered tissue beat spontaneously and showed contractile properties of native heart muscle including positive inotropic responses to calcium and isoprenaline. In group A recipient heart matched BioVADs were implanted into the corresponding healthy rats (n= 1 0), while in group B the spheric BioVADs were implanted (n= 10). 14 days after implantation hearts were explanted for histology. Histological analysis of recipient-matched BioVADs showed excellent coverage of the epicardial heart surface, while spheric BioVADs showed high number of micro and macro gaps. Furthermore direct alignment of the BioVADs and recipients hearts was measured in cross-sectioned histological compounds. In group B, a statistically higher number of areas with loss of contact between BioVADs and recipient hearts was seen compared to group A. Conclusion: We developed a novel casting technology using small animal MRI and 3D printing to generate artificial cardiac tissue that is perfectly matched to the recipient heart and exhibits structural and functional properties of native myocardium.
2( 52) DAP12 Expression By Lung-Resident Macrophages Mediates Pulmonary Ischemia Reperfusion Injury By Promoting Neutrophil Extravasation J. Spahn ,1 W. Li,1 A.C. Bribriesco,1 J. Liu,1 B. Zinselmeyer,2 H. Shen,3 S.L. Brody,2 D. Goldstein,3 A.S. Krupnick,1 A. Gelman,1 D. Kreisel.1 1Surgery, Washington University St. Louis, St. Louis, MO; 2Medicine, Washington University St. Louis, St. Louis, MO; 3Medicine, Yale University, New Haven, CT. Purpose: Neutrophils infiltrate lung grafts early after reperfusion and are important mediators of primary graft dysfunction. Immune pathways that regulate neutrophil recruitment to lungs during ischemia reperfusion injury remain poorly understood. The purpose of this study was to examine the role of DAP12, an adaptor molecule that regulates innate immune responses, in mediating ischemia reperfusion injury after lung transplantation. Methods: We used a mouse model of orthotopic vascularized lung transplantation. Grafts were assessed functionally (arterial blood gases), immunohistochemically, by quantitative PCR, flow cytometry and intravital two-photon imaging. Chemokine production by macrophages in vitro was assessed by ELISA. Results: We found that macrophages in mouse (qPCR and immunohistochemistry) and human (immunohistochemistry) lungs that have been subjected to cold ischemia express DAP12. Donor, but not recipient deficiency in DAP12 protected against pulmonary ischemia reperfusion injury as evidenced by improved graft function (WT-> WT, mean paO2= 1 80.8 +/-18.9, n= 1 4, DAP12KO-> W T, mean paO2= 410.8 +/-36.9, n= 8, WT-> DAP12KO mean paO2= 217.9 +/-55.9, n= 7 ). Compared to wild-type (WT) B6 cells, DAP12KO bone marrow-derived macrophages produced lower amounts of the neutrophil chemoattractant CXCL2 after in vitro stimulation (17 hours) with lysates derived from syngeneic B6 lung transplants (n= 2 ). Similarly, quantitative PCR of sorted donor macrophages 2 hours after reperfusion in vivo revealed significantly lower expression of CXCL2 when DAP12 was absent (n= 4 ). Intravital two-photon imaging using LysM-GFP neutrophil reporter mice as recipients demonstrated a transendothelial migration defect of neutrophils into DAP12KO lungs at 4 hours after reperfusion (percentage of extravascular neutrophils: WT-> LysM-GFP= 6 9.3%, n= 3 ; DAP12KO-> LysM-GFP= 2 5.6%, n= 3). Neutrophil entry into DAP12-deficient lungs was restored when WT bone-marrow derived macrophages (76.8%, n= 3) or recombinant CXCL2 (63.3%, n= 3) was administered into the donor bronchus prior to reperfusion. Conclusion: Our study has uncovered a previously unrecognized role for DAP12 expression in tissue-resident macrophages in mediating ischemia reperfusion injury after lung transplantation through regulation of neutrophil trafficking.