383: MyD88-Independent Signaling Pathway Is Involved with Lung Ischemic Preconditioning

383: MyD88-Independent Signaling Pathway Is Involved with Lung Ischemic Preconditioning

Abstracts 380 Non-Volume-Loaded Heart Provides a More Relevant Heterotopic Transplantation Model K.R. Tang-Quan,1,2 J. Bartos,2 T. Deuse,1,2 R.C. Robb...

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Abstracts 380 Non-Volume-Loaded Heart Provides a More Relevant Heterotopic Transplantation Model K.R. Tang-Quan,1,2 J. Bartos,2 T. Deuse,1,2 R.C. Robbins,2 H. Reichenspurner,1 S. Schrepfer.1,2 1University Heart Center Hamburg, Hamburg, Germany; 2Stanford University School of Medicine, Stanford, CA. Purpose: This study aimed to compare two techniques of heterotopic heart transplantations in rats. Our interest was focused on chronic allograft morphology, physiology, and immunology in the non-volume-loaded (NL) and volume-loaded (VL) model. Methods and Materials: Heterotopic heart transplantations were performed in the PVG-to-ACI rat model and followed over 90 days. Graft morphology was assessed by weight, histology after 90 days, and echocardiography on days 15, 30, 45, 60, and 90. Heart physiology was investigated by analysis of pulse pressure and maximum dP/dt using Langendorff perfusion. We observed the immunological response through the development of chronic allograft vasculopathy. Results: Ischemic time was significantly longer using the VL model compared to the NL model (p⬍0.001). Echocardiography revealed no significant differences in posterior wall thickness between the groups. The VL donor heart showed significantly higher weight, decreased pulse pressure, and decreased maximum dP/dt. Histological analysis showed a significant amount of luminal obliteration in the allogeneic transplantation groups (31-37%), but no difference in luminal obliteration between the two models (p⫽0.61). Remarkably, donor hearts in the VL model suffered from chronic myocardial ischemia, showing dilation and scarring on histology. Conclusions: Due to model design, the VL model suffers from dilated cardiomyopathy after 90 days. Despite the NL model not pumping blood volume, it has a shorter ischemic time and comparable immunological results to the VL. We conclude the NL model to be the more clinically relevant model in chronic heart transplantation studies. 381 The Effect of Gastro-Duodenal Contents on Tracheal Mucosa: There Is More to It than Acid V. Mertens,1 R. Farre,1 K. Blondeau,1 A. Pauwels,1 D. Sifrim,1,2 L. Dupont.3,4 1University Hospital Gasthuisberg, Leuven, Belgium; 2 Barts and The London School of Medicine and Dentistry, University of London, London, United Kingdom; 3Katholieke Universiteit Leuven, Leuven, Belgium; 4University Hospital Gasthuisberg, Leuven, Belgium. Purpose: Aspiration of gastric components (bile acids, pepsin, endotoxins) is common after lung transplantation and has been associated with the development of Bronchiolitis Obliterans Syndrome (BOS). However, the pathophysiological mechanism in the relationship between aspiration and development of BOS remains unclear. The aim of this study was to evaluate the effect of acid and non acidic solutions with or without gastro-duodenal contents on tracheal mucosa in an Ussing Chamber model. Methods and Materials: New Zealand rabbits were euthanized and segments of the whole trachea were mounted in Ussing Chambers for transepithelial mucosal resistance (TER). Substance P release was measured at the mucosal side as a marker of mucosal injury. The mucosal side was exposed for 20 min to a control solution (Krebs pH 7.4) or to test solutions containing deoxycholic acid (DC, 0.5-2mM), pepsin (0.4-1 mg/ml) or endotoxin (LPS) (1 ␮g/ml). Results: Exposure of the tracheal mucosa to acidic solutions (pH 1.2, 1.5 and 2), without gastric components, induced a significant drop in TER of 82, 73 and 55% respectively (N⫽4), suggestive of loss of tissue integrity. Less acidic solutions (pH⬍3) did not have an effect on the integrity of the tissue. However incubating the tissue with DC (0.5-2mM) at weakly acidic pH (pH ⬎ 3) significantly reduced the TER or integrity of the tissue, whereas pepsin (0.4-1 mg/ml) was not able to provoke a changes in TER at pH ⬎ 3 (N⫽4). 20 min exposure of the tracheal mucosa to DC (1.5mM, pH 7.4) or pepsin (1 mg/ml, pH 7.4) did not show an increase in SP at the mucosal side. However, exposure to LPS (1 ␮g/ml, pH 7.4) significantly increased SP release compared to the control solution. Conclusions: Not only acidic solutions (pH⬍3) but also weakly acidic

S127 solutions containing bile acids harm the integrity of the tracheal mucosa in vitro. Local SP release induced by endotoxins present in gastro-duodenal refluxate may further promote an inflammatory reaction. Further research is necessary to elucidate the pathogenesis of aspiration induced lung damage. 382 Reduced Transplant Arteriosclerosis after Treatment with MMF and MMF in Combination with Ganciclovir in a Mouse Aortic Transplant Model J. Hoffmann, M. Bohm, S. Abele-Ohl, M. Weyand, S.M. Ensminger. Friedrich-Alexander-University of Erlangen-Nu¨rnberg, Erlangen, Germany. Purpose: The aim of this study was to investigate whether Ganciclovir and Mycophenolate Mofetil (MMF), alone and in combination, have a beneficial influence on the development of transplant arteriosclerosis (TxA). Methods and Materials: Fully allogeneic C57BL/6 (H2b) donor aortas were transplanted into CBA (H2k) recipients. Recipient mice were treated with a single medication of MMF (100mg/kg or 300mg/kg) or a combination of MMF and Ganciclovir (10mg/kg Ganciclovir and 100mg/kg MMF or 72mg/kg Ganciclovir and 300mg/kg MMF) per day. Grafts were analysed by histology and morphometry on day 30 after transplantation. A potential effect of MMF or Ganciclovir on Tregs was investigated by FACS analysis. Results: Two doses of 150mg/kg MMF per day significantly reduced the development of TxA compared to untreated controls (intima proliferation of 23% ⫹/⫺ 8% vs. 68% ⫹/⫺ 7% [control]). 50mg/kg of MMF did not have any effect on the development of TxA. Strikingly, combination of Ganciclovir and low-dose MMF reduced the formation of TxA (intima proliferation of 27% ⫹/⫺ 2% vs. 68% ⫹/⫺ 7% [control]). The high-dose combination group (300mg MMF/72mg Ganciclovir) showed an intima proliferation of 24% ⫹/⫺ 4%. FACS analysis of the spleen within this group revealed 12,73% CD4⫹ cells with 14,70% CD4⫹CD25⫹FoxP3⫹ cells. As compared to 17,5% CD4⫹ cells with 11,1% CD4⫹CD25⫹FoxP3⫹ cells in the control group. Single treatment with MMF (300mg) showed 10,55% CD4⫹ cells and 10,87% CD4⫹CD25⫹FoxP3⫹ cells within the analysed spleens. Conclusions: Our results demonstrate a dose-dependent positive effect of MMF on the development of TxA and a synergistic effect of Ganciclovir in combination with MMF. 383 MyD88-Independent Signaling Pathway Is Involved with Lung Ischemic Preconditioning A. Shimamoto, Y. Yajima, S. Shomura, M. Takao, H. Shimpo. Mie University Graduate School of Medicine, Tsu, Japan. Purpose: We previously reported that the absence of Toll-like receptor 4 (TLR4) in TLR4-knockout mice (KO) attenuates the markers of an inflammatory response including NF-␬B activation and substantially reduces lung ischemia-reperfusion (I/R) injury (LIRI). TLR4 signaling including NF-␬B is also involved in ischemic preconditioning (IPC), however its mechanism is still unclear. Recently, MyD88-independent pathway through TRIF, which is another MyD88-dependent pathway of TLR4 signaling through NF-␬B, is discovered and suggested to be concerned with organ preservation. In the present study, we evaluated how TLR4 signaling regulates NF-␬B causing to LIRI or IPC. Methods and Materials: Wild-type (WT, C57BL/6J) mice were compared to TLR4-, MyD88-, and TRIF-KO underwent 60 minutes of ischemia of their left lungs followed by 180 minutes of reperfusion (I/R alone) or IPC with six cycles of 5 minutes ischemia and 5 minutes reperfusion prior to I/R. Response to injury was quantified by tissue MPO activity, vascular permeability, and leukocyte and inflammatory mediator accumulation in BAL expression. Lung homogenates were also analyzed for activation of NF-␬B. Results: The absence of TLR4 and MyD88 resulted in less NF-␬B activation (P⬍.01), and the development of a significant smaller LIRI which quantified by vascular permeability (P⬍.001), MPO activity (P⬍.002), and

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The Journal of Heart and Lung Transplantation, Vol 29, No 2S, February 2010

BAL components after I/R alone. Compared to I/R alone, IPC decreased LIRI in WT but increased LIRI in TLR4 KO and TRIF KO; MyD88 KO was unchanged. Compared to sham controls, IPC in WT led to significant increment of NF-␬B activation (P⫽.008). Similar increases were not observed in TLR4 KO and TRIF KO. Conclusions: We provide evidence for a protective role of the innate immune system in lung IPC. These data indicate that IPC is mediated a MyD88-independent pathway, leading to NF-␬B activation and protection against LIRI. We conclude that an activation of MyD88 or TRIF, which are associate proteins of TLR4, would regulate a role of NF-␬B to injure or preserve lung function. 384 Egfl7 Prevents Coronary Endothelial ICAM-1 Expression in Response to Calcineurin Inhibition M.V. Badiwala, L.C. Tumiati, A. Ghashghai, J.M. Joseph, V. Rao. Toronto General Hospital, Toronto, ON, Canada. Purpose: Expression of intercellular adhesion molecule-1 (ICAM-1) is a key step in the inflammatory cascade that occurs with endothelial injury. We determined the effect of Egfl7 on coronary endothelial ICAM-1 expression in response to tacrolimus and cyclosporine. Furthermore, we examined whether the effect of Egfl7 is mediated by the NOTCH signaling pathway. Methods and Materials: HCAEC (n⫽4 to 6) were incubated with tacrolimus (10-100 ng/mL), CyA (0.1-10 mcg/mL) or 0.01% DMSO (control) in the presence or absence of Egfl7 (100 ng/mL) or the NOTCH receptor activator Jagged1 (200 ng/mL) for 48h. Expression of ICAM-1 was determined by flow cytometry. Results: Incubation with tacrolimus or CyA upregulated ICAM-1 expression, which was significantly attenuated by co-administration of Egfl7 (Table 1). Co-incubation with Jagged1 (200ng/mL) alone had no effect on ICAM-1 expression, however, it attenuated the ICAM-1 suppressive effect of Egfl7 when administered in combination with CyA (193⫾3% vs. 148⫾5%, p⫽0.006). Table 1 ICAM-1 Expression % of Control ICAM-1 Expression

Group

Without Egfl7 (p⫽compared to control)

With Egfl7 (100ng/mL) (p⫽ compared to group without Egfl7)

Control (0.01% DMSO) Tacrolimus 10ng/mL Tacrolimus 100ng/mL CyA 0.1 mcg/mL CyA 1.0 mcg/mL CyA 10 mcg/mL

100⫾3% 123⫾8%, p⬎0.05 304⫾21%, p⬍0.001 115⫾5%, p⬎0.05 126⫾2%, p⬍0.001 215⫾13%, p⬍0.001

65⫾3%, p⬍0.0001 105⫾2%, p⫽0.009 215⫾15%, p⫽0.007 92⫾6%, p⫽0.01 111⫾12%, p⫽0.04 148⫾5%, p⫽0.004

Conclusions: Our study reveals the novel observation that Egfl7 inhibits coronary artery endothelial cell expression of ICAM-1 subsequent to CNI with tacrolimus or cyclosporine. Mechanistically, Egfl7 may inhibit NOTCH receptor activation. Egfl7 may be protective against CNI injury incurred after transplantation and thus may modulate the events that lead to the development of cardiac allograft vasculopathy. 385 Expression of a Stablized Hypoxia Inducible Factor-1␣ Reduces Cardiomyocyte Apoptosis and Prevents Cardiac Allograft Vasculopathy M.A.I. Keranen,1 A.I. Nykanen,1 R. Krebs,1 K. Pajusola,2 K. Alitalo,2 K.B. Lemstrom.1,3 1University of Helsinki and Helsinki University Central Hospital, Helsinki, Finland; 2Biomedicum Helsinki, University of Helsinki and Helsinki University Central Hospital, Helsinki, Finland; 3 University of Helsinki and Helsinki University Central Hospital, Helsinki, Finland.

Purpose: Hypoxia inducible factor-1 (HIF-1), a key transcription factor in angiogenesis, affects a wide range of adaptive cell functions, and potentially also the development of cardiac allograft vasculopathy (CAV). Methods and Materials: Heterotopic cardiac allograft transplantations were performed between major MHC-mismatched DA rats (donors) and WF rats (recipients). In the acute rejection model, no immunosuppression was used. In the chronic rejection model, the recipients were given cyclosporine A 2 mg/kg for the first 7 days and 1 mg/kg/day thereafter to prevent acute rejection. Syngeneic (DA to DA) transplants served as controls. Endogenous HIF-1␣ level during acute and chronic rejection was analyzed with immunohistochemistry and qPCR. In addition, we examined the effect of exogenous HIF-1␣ in chronically rejecting rat cardiac allografts by intracoronary adeno-associated virus-mediated gene transfer. Results: Acute and chronic rejection significantly induced HIF-1␣ mRNA in cardiac allografts when compared to syngeneic controls. Immunoreactivity of HIF-1␣ was localized to vascular smooth muscle cells, vascular endothelial cells, postcapillary venules and graft-infiltrating mononuclear inflammatory cells of the allograft, while cardiomyocytes were unaffected. A linear correlation was observed between the progression of CAV and HIF-1␣ immunoreactivity in postcapillary venules and mononuclear inflammatory cells of the allograft. Gene transfer of stabilized HIF-1␣ enhanced cardiomyocyte HIF-1␣ production, prevented cardiomyocyte apoptosis and significantly reduced CAV in chronically rejecting cardiac allograts. Conclusions: We found acute and chronic cardiac allograft rejection as potent stimuli leading to HIF-1␣ stabilization in vascular and inflammatory cells. Furthermore, cardiomyocyte-targeted HIF-1␣ gene transfer inhibited the progression of CAV, suggesting that HIF-1␣ overexpression could provide therapeutic benefit in cardiac allografts. 386 Contribution of Pre-Existing Vascular Disease to Allograft Vasculopathy in Mice A. Zaki, J. Jordan, G. Hirsch, B. Ross, T. Lee. Dalhousie University, Halifax, NS, Canada. Purpose: Allograft vasculopathy (AV) is characterized by a progressive vascular lesion in the coronary arteries of transplant patients.This lesion is resistant to current immunosuppressive therapies.Previous rodent AV models have shown that this lesion is recipient, however human studies suggest it is chimeric, with donor and recipient elements.We believe that this discrepancy is because, in rodent models, the neointima is a de novo lesion that is developed by the recruitment of recipient cells.However, in human AV, we believe that the lesion also contains elements of pre-existing donor vasculopathy. Methods and Materials: To confirm the recipient nature of the lesion in the presence of Cyclosporine A (CyA;50mg/kg/d), we used B6 Green Fluorescent Protein (GFP) mice recipients of C3H aortic grafts.To determine the role of pre-existing disease on AV, we used apolipoprotein-E-deficient (ApoE⫺/⫺) mice as donors.Aortic sections from ApoE⫺/⫺ B6 mice were transplanted into C3H’s and, after 10wk, the lesions were assessed for AV and cellular infiltration. Results: 97% of the neointimal lesion in the GFP experimental group was positive for green fluorescence compared to 0% in controls, demonstrating the recipient nature of the neointima.In our ApoE⫺/⫺ model, we found that donor pre-existing lesions were retained in the graft in the presence of CyA.Furthermore, we determined that the de novo AV lesion overlaid the pre-existing lesion, forming two discrete lesions.The presence of the preexisting lesion did not affect the progression of the de novo AV lesion; however, it increased the overall lesion size (121000 ␮m ⫾24340␮m vs 91390 ␮m ⫾ 11080␮m in controls) and increased the percent luminal narrowing (65% ⫾7.2% vs 49% ⫾4.5% in controls). Conclusions: Even in the presence of calcineurin inhibition, the neointimal lesion in mouse models is recipient in origin.The effect of the pre-existing lesions on the overall intimal area may explain the data in the literature suggesting poor outcomes associated with transplantation of cardiac grafts with mild to moderate pre-existing atherosclerosis.