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130: Versican Expression Induces Tissue Remodeling
The Journal of Heart and Lung Transplantation Volume 28, Number 2S
Purpose: Polyclonal antithymocyte globulins (ATGs) are agents employed for induction of immunosuppression after organ transplantation. We have previously shown that ATGs influence the expression of adhesion molecules in an animal model. We postulated that ATGs would have an isolated effect on endothelial cells (EC), reducing the leukocyte transmigration and the extent of endothelial response. Methods and Materials: Cells from an immortalized human microvascular endothelial cell line (HMEC) were incubated with ATG (ATG-S ©, Fresenius Biotech, Germany) for 2 hours on collagencoated polytetrafluorethylene (PTFE) filters. Endothelial transmigration of peripheral blood mononuclear cells (PMBC) was assessed in different wells (n⫽20 in 4 independent experiments). Expression of ICAM-1 and MHC class I molecules on HMEC was studied by flow-cytometry. Unspecific rabbit IgG was used as control. Statistical analysis was done with the 2-tailed Student’s t-test. Results: PMBC transendothelial migration was significantly reduced after ATG treatment of HMEC as compared to untreated controls (1.55E⫹05 ⫾ 1.00E⫹04 vs. 6.85E⫹05 ⫾ 1.87E⫹05; p⬍0.05). Incubation with rabbit IgG showed no significant differences. Incubation of EC with ATG significantly reduced the surface expression of ICAM-1 (73.3 ⫾ 4.1 % vs. 33.4 % ⫾ 4.1 positive cells, p⬍0.001) and MHC class I (100.0 ⫾ 3.5 % vs. 59.5 ⫾ 4.6 %, p⬍0.001) on HMEC. Conclusions: Incubation of isolated EC with ATGs prevented the transendothelial migration of PMBC. Furthermore, a decrease of the expression of endothelial adhesion molecules could be assessed. In addition to reduced endothelial activity, down-regulation of adhesion molecules by ATG may decrease graft cell infiltration after solid organ transplantation. 130 Versican Expression Induces Tissue Remodeling J.M. Carthy, S. Boroomand, M. Rahmani, D. Knight, B.M. McManus The University of British Columbia/The James Hogg iCAPTURE Centre, Vancouver, BC, Canada Purpose: Versican is a chondroitin sulfate proteoglycan found in the extracellular matrix (ECM) of many tissues and is a major component that accumulates in lesions of cardiac allograft vasculopathy. Versican is considered pro-atherogenic and central to vascular injury and repair events because of its ability to trap cholesterol-rich lipoproteins, in addition to its impact on cell adhesion, survival, proliferation, and migration. The current study evaluated the hypothesis that versican modifies tissue remodeling by altering fibroblast-mediated contraction of the ECM. Methods and Materials: We have cloned human versican and set up an inducible (doxycycline controlled) expression system in mouse embryonic fibroblasts. Cells were cultured in 3D native type I collagen gels in the presence or absence of versican overexpression, and contraction was followed for a period of 24 hours. Results: Results suggest versican significantly increased fibroblast contraction of the collagen gels (27.9 ⫾ 1.2% vs 64.5 ⫾ 1.4% of initial gel area, p⬍0.01). Immunohistochemistry and confocal imaging of these gels demonstrated a marked change in cell phenotype: versican expressing fibroblasts exhibited cellular protrusions that were associated with smooth muscle alpha-actin (SMA) positive stress fibres (a marker of myofibroblast differentiation), while non-induced cells were rounded and negative for SMA. Further, versican appeared to form a pericellular coat around these cellular protrusions suggesting it may be influencing cell receptor-to-matrix interactions. In support of this idea, Western blotting demonstrated that versican overexpression dramatically increased the expression of beta 1 integrin. Inhibition of versican expression by siRNA completely reversed the versican-induced change in cell phenotype. Conclusions: Taken together, our data suggests versican induces myofibroblast differentiation during tissue remodeling. If versican
Abstracts
S111
also promotes tissue remodeling in vivo, the relative amount of versican produced at sites of injury may be critical in regulating vessel wall geometry (shrinkage) after heart transplantation. 131 The Association between Killer Immunoglobulin-Like Receptors (KIR) Genotype Profile and Primary CMV Infection in CMV Dⴙ/R- Heart and Lung Transplant Recipients S. Hemachandra1, M. Askar2, A. Zhang2, D. Thomas2, D. Pidwell2, N.K. Shrestha1, D. van Duin1, R.K. Avery1 1Cleveland Clinic Foundation, Cleveland, OH; 2Cleveland Clinic Foundation, Cleveland, OH Purpose: CMV donor positive/recipient negative (D⫹/R-) recipients are at high risk for severe CMV disease, but why only some D⫹/Rpatients become viremic or develop severe disease is not known. The inheritance of multiple activating Killer Immunoglobulin-Like Receptors (aKIR) genes has been reported to influence immune control of CMV in kidney transplant recipients. We investigated the relationship between recipient KIR genotype and CMV disease in D⫹/R- thoracic organ transplant recipients. Methods and Materials: A retrospective analysis was performed on all D⫹/R- heart and lung transplant recipients from 1999-2005. KIR genotype was determined on stored genomic samples by PCR sequencing specific oligonucleotide probe. Individuals carrying only KIR3DL3, 2DL3-2DL1- 2DP1-3DP1-2DL4-3DL1-2DS4-3DL2 were considered as carrying 2 copies of group A haplotypes, and designated ‘B-’. The remainders were designated ‘B⫹’. Patients were also grouped based on the number of aKIRs that their genotypes contained. Outcomes measured were: viremia, high level CMV viremia (⬎100,000 copies/ml), tissue-invasive CMV disease, and recurrent CMV viremia. Results: Of 46 recipients (12 heart and 34 lung), 13 (28%) were B-, and 33 (72%) were B⫹. 77% in the B- group vs. 45% of B⫹ patients had CMV viremia (p⫽0.004). There were no significant differences for the other evaluated outcomes in the B⫹ and B- groups. Conclusions: These findings suggest the presence of an association between KIR genotype and the occurrence and severity of primary CMV infection in SOT D⫹/R- recipients. Further study is required to confirm these findings and to optimally stratify KIR genotypes to predict clinical outcomes. Heart and Lung CMV outcomes in 1-3 vs. 4-6 aKIR genes Viremia CMV Vl ⬎100K Recurrent CMV Invasive CMV
1-3 aKIR
4-6 aKIR
p-value
64% 11% 54% 18%
39% 11% 22% 22%
0.13 1.0 0.06 0.72
Lung Transplant Outcomes Viremia Recurrent CMV
1-3aKIR
4-6aKIR
p-value
68% 57%
33% 17%
0.08 0.03
132 The Role of Mast Cells in Acute and Chronic Cardiac Allograft Rejection S. Itoh1, S. Nakae2, J. Velotta1, H. Kosuge1, S. Schrepter1, A. Connolly3, M. Tsai3, H. Adachi4, S.J. Galli3, R.C. Robbins1, M.P. Fischbein1 1Stanford University School of Medicine, Stanford; 2 University of Tokyo, Tokyo, Japan; 3Stanford University School of Medicine, Stanford; 4Saitama Medical Center, Jichi Medical School, Saitama, Japan
Purpose: Polyclonal antithymocyte globulins (ATGs) are agents employed for induction of immunosuppression after organ transplantation. We have previously shown that ATGs influence the expression of adhesion molecules in an animal model. We postulated that ATGs would have an isolated effect on endothelial cells (EC), reducing the leukocyte transmigration and the extent of endothelial response. Methods and Materials: Cells from an immortalized human microvascular endothelial cell line (HMEC) were incubated with ATG (ATG-S ©, Fresenius Biotech, Germany) for 2 hours on collagencoated polytetrafluorethylene (PTFE) filters. Endothelial transmigration of peripheral blood mononuclear cells (PMBC) was assessed in different wells (n⫽20 in 4 independent experiments). Expression of ICAM-1 and MHC class I molecules on HMEC was studied by flow-cytometry. Unspecific rabbit IgG was used as control. Statistical analysis was done with the 2-tailed Student’s t-test. Results: PMBC transendothelial migration was significantly reduced after ATG treatment of HMEC as compared to untreated controls (1.55E⫹05 ⫾ 1.00E⫹04 vs. 6.85E⫹05 ⫾ 1.87E⫹05; p⬍0.05). Incubation with rabbit IgG showed no significant differences. Incubation of EC with ATG significantly reduced the surface expression of ICAM-1 (73.3 ⫾ 4.1 % vs. 33.4 % ⫾ 4.1 positive cells, p⬍0.001) and MHC class I (100.0 ⫾ 3.5 % vs. 59.5 ⫾ 4.6 %, p⬍0.001) on HMEC. Conclusions: Incubation of isolated EC with ATGs prevented the transendothelial migration of PMBC. Furthermore, a decrease of the expression of endothelial adhesion molecules could be assessed. In addition to reduced endothelial activity, down-regulation of adhesion molecules by ATG may decrease graft cell infiltration after solid organ transplantation. 130 Versican Expression Induces Tissue Remodeling J.M. Carthy, S. Boroomand, M. Rahmani, D. Knight, B.M. McManus The University of British Columbia/The James Hogg iCAPTURE Centre, Vancouver, BC, Canada Purpose: Versican is a chondroitin sulfate proteoglycan found in the extracellular matrix (ECM) of many tissues and is a major component that accumulates in lesions of cardiac allograft vasculopathy. Versican is considered pro-atherogenic and central to vascular injury and repair events because of its ability to trap cholesterol-rich lipoproteins, in addition to its impact on cell adhesion, survival, proliferation, and migration. The current study evaluated the hypothesis that versican modifies tissue remodeling by altering fibroblast-mediated contraction of the ECM. Methods and Materials: We have cloned human versican and set up an inducible (doxycycline controlled) expression system in mouse embryonic fibroblasts. Cells were cultured in 3D native type I collagen gels in the presence or absence of versican overexpression, and contraction was followed for a period of 24 hours. Results: Results suggest versican significantly increased fibroblast contraction of the collagen gels (27.9 ⫾ 1.2% vs 64.5 ⫾ 1.4% of initial gel area, p⬍0.01). Immunohistochemistry and confocal imaging of these gels demonstrated a marked change in cell phenotype: versican expressing fibroblasts exhibited cellular protrusions that were associated with smooth muscle alpha-actin (SMA) positive stress fibres (a marker of myofibroblast differentiation), while non-induced cells were rounded and negative for SMA. Further, versican appeared to form a pericellular coat around these cellular protrusions suggesting it may be influencing cell receptor-to-matrix interactions. In support of this idea, Western blotting demonstrated that versican overexpression dramatically increased the expression of beta 1 integrin. Inhibition of versican expression by siRNA completely reversed the versican-induced change in cell phenotype. Conclusions: Taken together, our data suggests versican induces myofibroblast differentiation during tissue remodeling. If versican
Abstracts
S111
also promotes tissue remodeling in vivo, the relative amount of versican produced at sites of injury may be critical in regulating vessel wall geometry (shrinkage) after heart transplantation. 131 The Association between Killer Immunoglobulin-Like Receptors (KIR) Genotype Profile and Primary CMV Infection in CMV Dⴙ/R- Heart and Lung Transplant Recipients S. Hemachandra1, M. Askar2, A. Zhang2, D. Thomas2, D. Pidwell2, N.K. Shrestha1, D. van Duin1, R.K. Avery1 1Cleveland Clinic Foundation, Cleveland, OH; 2Cleveland Clinic Foundation, Cleveland, OH Purpose: CMV donor positive/recipient negative (D⫹/R-) recipients are at high risk for severe CMV disease, but why only some D⫹/Rpatients become viremic or develop severe disease is not known. The inheritance of multiple activating Killer Immunoglobulin-Like Receptors (aKIR) genes has been reported to influence immune control of CMV in kidney transplant recipients. We investigated the relationship between recipient KIR genotype and CMV disease in D⫹/R- thoracic organ transplant recipients. Methods and Materials: A retrospective analysis was performed on all D⫹/R- heart and lung transplant recipients from 1999-2005. KIR genotype was determined on stored genomic samples by PCR sequencing specific oligonucleotide probe. Individuals carrying only KIR3DL3, 2DL3-2DL1- 2DP1-3DP1-2DL4-3DL1-2DS4-3DL2 were considered as carrying 2 copies of group A haplotypes, and designated ‘B-’. The remainders were designated ‘B⫹’. Patients were also grouped based on the number of aKIRs that their genotypes contained. Outcomes measured were: viremia, high level CMV viremia (⬎100,000 copies/ml), tissue-invasive CMV disease, and recurrent CMV viremia. Results: Of 46 recipients (12 heart and 34 lung), 13 (28%) were B-, and 33 (72%) were B⫹. 77% in the B- group vs. 45% of B⫹ patients had CMV viremia (p⫽0.004). There were no significant differences for the other evaluated outcomes in the B⫹ and B- groups. Conclusions: These findings suggest the presence of an association between KIR genotype and the occurrence and severity of primary CMV infection in SOT D⫹/R- recipients. Further study is required to confirm these findings and to optimally stratify KIR genotypes to predict clinical outcomes. Heart and Lung CMV outcomes in 1-3 vs. 4-6 aKIR genes Viremia CMV Vl ⬎100K Recurrent CMV Invasive CMV
1-3 aKIR
4-6 aKIR
p-value
64% 11% 54% 18%
39% 11% 22% 22%
0.13 1.0 0.06 0.72
Lung Transplant Outcomes Viremia Recurrent CMV
1-3aKIR
4-6aKIR
p-value
68% 57%
33% 17%
0.08 0.03
132 The Role of Mast Cells in Acute and Chronic Cardiac Allograft Rejection S. Itoh1, S. Nakae2, J. Velotta1, H. Kosuge1, S. Schrepter1, A. Connolly3, M. Tsai3, H. Adachi4, S.J. Galli3, R.C. Robbins1, M.P. Fischbein1 1Stanford University School of Medicine, Stanford; 2 University of Tokyo, Tokyo, Japan; 3Stanford University School of Medicine, Stanford; 4Saitama Medical Center, Jichi Medical School, Saitama, Japan