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ACTIVATION OF CALCIUM-INDEPENDENT PHOSPHOLIPASE A2 IN TRYPTASE-STIMULATED HUMAN BLADDER ENDOTHELIAL CELLS
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Poster Abstracts / Cardiovascular Pathology 13 (2004) S80–S138
P307
P309
COMPOSITION AND REGULATION OF THE PECAM-CONTAINING RECYCLING MEMBRANE COMPARTMENT IN ENDOTHELIAL CELLS. Zahra Mamdouh, Xia Chen, William A. Muller. Weill Medical College of Cornell University, Department of Pathology Room C320, 1300 York Avenue. New York NY 10021.
REGULATION AND IMPLICATION IN MIGRATION OF MEMBRANE TYPE-1 MATRIX METALLOPROTEINASE IN HUMAN MONOCYTES. Salomo´n Matı´as-Roma´n, Beatriz G. Ga´lvez, Laura Genı´s, Alicia G. Arroyo. Centro de Investigaciones Biolo´gicas, Spain.
PECAM-1, a glycoprotein expressed on most leukocytes and on endothelial cells (EC) at the cell border, is a key molecule controlling diapedesis. We have recently shown that PECAM-1 enters an intracellular membrane compartment connected to the surface of human umbilical vein endothelial cells (HUVEC). Trafficking of PECAM-1 between this compartment and EC surface occurs evenly along the junction in resting cells. However, during diapedesis, recycling PECAM-1 is targeted preferentially to the zone of the junction surrounding the migrating monocyte. We also found that this compartment is novel, distinct from the endocytic recycling compartment and from caveolae. Furthermore, electron microscopy of postcapillary venules recovered from mice intravenously injected with anti-mouse PECAM-1 showed intracellular structures containing PECAM-1 similar to those we previously described in HUVEC. We investigated whether other junctional molecules were also present in the PECAM-containing membrane compartment. CD99 is in these structures while VE-cadherin is not. We also show that treatment of endothelial cells with demecolcine, a drug that blocks microtubule polymerization, alters the constitutive and targeted recycling of PECAM-1. This treatment also selectively blocks monocyte transendothelial migration by 80% without interfering with their ability to adhere to and crawl on the endothelial cell surface. This implies a role for microtubules in maintaining the structure or the function of this PECAM-containing compartment.
Recruitment of monocytes to tissues is a crucial event in the inflammatory response, not yet completely understood. The metalloproteinase MT1MMP is involved in the degradation of extracellular matrix ( ECM ), and in this regard, its action is important for the migration of different cell types. We demonstrate in this work that binding of human monocytes to fibronectin ( FN ), upregulates the expression of MT1-MMP, through the interaction with integrins a4 and a5. An increase in MT1-MMP is also observed after attachment of monocytes to either resting or activated human endothelial cells. In fact, interaction of monocytes with the endothelial ligands ICAM-1 or VCAM-1 also results in an induction of MT1-MMP expression. Moreover, the increase in MT1-MMP expression correlates with an upregulation of its fibrinolytic activity upon binding of monocytes to either FN or endothelial ligands. In addition, MT1-MMP is located at protrusive plasma membrane structures of migratory human monocytes. Finally, a functional role for MT1-MMP is demonstrated by inhibition of monocyte migration on FN with anti-MT1-MMP mAbs. All these data show that MT1-MMP is regulated by the ECM and the endothelium in human monocytes , and it plays an important role during their migration. Grant SAF2002-00068 from Ministerio de Ciencia y Tecnologı´a (Spain)
supported by NIH grant HL46849 to WAM P310
P308 DISSECTION OF THE ROLES OF ENDOTHELIAL ADHESION MOLECULES AND CHEMOKINES IN RECRUITMENT OF CD4+ T CELLS UNDER CONDITIONS OF PHYSIOLOGICAL FLOW. Thomas Manes, Jordan S. Pober, Martin S. Kluger. Yale University School of Medicine. TNF-treated human umbilical vein endothelial cell ( HUVEC ) monolayers overlaid with the chemokine SDF-1 are able to bind and stimulate the transendothelial migration ( TEM ) of CD4+T cells under conditions of physiologically relevant flow (1 dyne/cm2). Antibody blocking experiments and genetic deletion of specific TNF-induced adhesion molecules, such as E-selectin, ICAM-1, and VCAM-1, have identified roles for these proteins in T cell recruitment when expressed on inflamed endothelium, but it is not known if adhesion molecule expression is sufficient to account for the effects of TNF. To address this question, we prepared retrovirally transduced HUVEC that constitutively (in the absence of cytokine) express E-selectin, ICAM-1, and VCAM-1, either in pairwise combinations or in triple combination. We find that triple adhesion molecule expressing cells are able to support T cell TEM equally as well as TNF-treated EC. Double expressers performed less well and no individual adhesion molecule appeared to be indispensable, although favoring of specific T cell subsets may occur. In the absence of SDF-1, neither TNF-treated nor adhesion molecule-transduced HUVEC were able to stimulate TEM. These observations suggest that, in the presence of chemokine, adhesion molecule expression can largely account for the pro-inflammatory effects of TNF on T cell recruitment by EC and that there is overlap in the functions of Eselectin, ICAM-1, and VCAM-1 expressed on EC.
ACTIVATION OF CALCIUM-INDEPENDENT PHOSPHOLIPASE A2 IN TRYPTASE-STIMULATED HUMAN BLADDER ENDOTHELIAL CELLS. Maureen Meyer, Pamela Kell, Alice Rickard, Jane McHowat. Saint Louis University, Saint Louis, USA. Interstitial cystitis is an inflammatory bladder condition with unknown etiology, although a pathophysiologic role for mast cells has been implicated. We hypothesized that mast cell tryptase may contribute to the exacerbation of inflammation by activating the protease-activated receptor-2 (PAR-2) on human bladder microvascular endothelial cells (HBMEC), resulting in activation of phospholipase A2 (PLA2) and increased production of inflammatory phospholipid metabolites. Incubation of confluent monolayers of HBMEC with tryptase (0.2 to 200 ng/ml for intervals up to 60 mins) resulted in a dose- and time-dependent increase in platelet-activating factor (PAF) production and release of arachidonic acid, prostacyclin and prostaglandin E2. Pretreatment of HBMEC with the calcium-independent PLA2 (iPLA2) inhibitor bromoenol lactone ( BEL, 5 microM, 10 mins) resulted in complete inhibition of tryptase-stimulated production of phospholipid metabolites, suggesting that tryptase stimulates increased iPLA2 activity in HBMEC. Tryptase-simulated increases in PAF production were accompanied by increased expression of P-selectin on the HBMEC cell surface and resulted in a significant increase in adherence of neutrophils to the HBMEC monolayer. Pretreatment of HBMEC with methyl arachidonyl fluorophosphonate (MAFP, 5 microM, 10 mins, a cytosolic PLA2 inhibitor which also inhibits PAF acetylhydrolase activity) resulted in a significant potentiation of tryptase-stimulated PAF production, cell surface expression of P-selectin and neutrophil adherence. These data indicate that tryptase stimulation of HBMEC results in activation of iPLA2, resulting in increased membrane phospholipid hydrolysis and accumulation of several inflammatory mediators. In particular, the increase in cell surface expression of P-selectin and the increased neutrophil adherence to the endothelial cell monolayer following tryptase incubation
Poster Abstracts / Cardiovascular Pathology 13 (2004) S80–S138 suggests that this protease may play an important role in propagation of the inflammatory response. National Institute of Diabetes and Digestive and Kidney Diseases DK066119
P311 TOLL-LIKE RECEPTOR 2 ACTIVATION OF MONOCYTES DECREASES ADHESION TO ENDOTHELIAL-LIKE CELLS UNDER FLOW CONDITIONS. Manon M Oude Nijhuis, Laurien H Ulfman, Arjan H Schoneveld, Dominique PV de Kleijn, Nienke I Sluis, Gerard Pasterkamp. Experimental Cardiology Laboratory, University Medical Center Utrecht, Utrecht, Netherlands, Department of Pulmonary Diseases, University Medical Center Utrecht, Utrecht, Netherlands. Background: Atherosclerosis is associated with an intense inflammatory response. During this response mediators like chemokines and cytokines are able to recruit leukocytes and macrophages to the site of injury. Previously, we demonstrated that peptidoglycan ( PGN ), a Toll-like receptor 2 ( TLR-2 ) ligand, is present in unstable atherosclerotic lesions hiding inflammatory cells. The presence of peptidoglycan was mainly observed in macrophage-rich regions. In addition, we demonstrated a significant relation between IgM antibody levels against this TLR-2 ligand and carotid intima-media thickness. In the present study, we determined the effect of TLR-2 ligands on monocyte adhesion to endothelial-like cell layers under flow conditions. Methods: The TLR-2 ligands used are purified Staphylococcus aureus PGN and synthetic Pam3Cys. On monocytes, activated by these TLR-2 ligands, the expression of adhesion molecules CD18, CD11b and CD62L was measured by flow cytometry. Furthermore, an in vitro flow chamber model was used to study the interaction between PGN- and Pam3Cys- activated monocytes to endothelial-like cells (Chinese Hamster Ovary cells (CHO cells) expressing E-selectin, and L-cells expressing E-selectin and ICAM-1). The role of L-selectin on adhesion of the monocytes to the endothelial-like cells was evaluated by the use of a blocking antibody ( Dreg56 ). Results: Higher levels of the monocyte receptors CD18 and CD11b were expressed on monocytes activated with the TLR-2 ligands compared to control monocytes (CD18; 137.0+/-22.4 versus 79.9+/ 11.0, CD11b; 221.2+/ 57.8 versus 132.1+/ 21.9 for Pam3Cys and CD18; 126.9+/ 22.7 versus 91.4+/ 18.2, CD11b; 185.5+/ 32.5 versus 94.2+/ 16.7 for PGN), while L-selectin (CD62L) was shed from the surface after monocyte activation (10.3+/ 2.8 versus 38.3+/ 6.8 for Pam3Cys and 23.8+/ 10.4 versus 47.2+/ 5.3 for PGN). TLR2 activation resulted in a significantly reduced number of adherent monocytes to both the CHO cells and the Lcells compared to nonactivated control monocytes. This suggests that Lselectin shedding results in a decrease in monocyte tethering to Eselectinexpressing endothelial cells. Indeed, in the presence of an Lselectin blocking antibody adherence of control monocytes dropped to values similar to monocyte adhesion after TLR2 activation. Conclusion: These results show that monocytes increase the expression of adhesion receptors on their surface and shed Lselectin upon TLR2 activation. Surprisingly, however, the adhesion of monocytes to endotheliallike cells is decreased after TLR2 activation probably due to the Lselectin shedding. Future studies will evaluate the role of CD11b, CD18 on TLR2 stimulated monocytes in the shear resistance to endothelial cells. This might reveal the mechanism by which peptidoglycan enters the atherosclerotic plaque.
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P312 THE FIBRIN-DERIVED PEPTIDE BB15-42 REDUCES MYOCARDIAL REPERFUSION INJURY. Peter Petzelbauer, Peter Friedl, Marion Gro¨ger, Paula Zacharowski, Klaus Wolff, Kai Zacharowski. Department of Dermatology, Division of General Dermatology, University of Vienna, Medical School, A-1090 Vienna, Austria, Department of Dermatology, Division of General Dermatology, University of Vienna, Medical School, A-1090 Vienna, Austria, Molecular Cardioprotection and Inflammation Group, Department of Anaesthesiology, University Hospital of Du¨sseldorf, D-40225, Du¨sseldorf, Germany. VE-cadherin is typically expressed within junctional regions of neighbouring endothelial cells, where it forms homotypic adhesion complexes. Recently the N-terminal sequence of the fibrin beta-chain was identified as a novel binding partner for VE-cadherin. Unexpectedly, by binding to endothelial VE-cadherin, fibrin fragments induce transmigration of T cells, monocytes and neutrophils across monolayers of endothelial cells in culture. Peptide Bbeta15-42, which matches the N-terminal sequence of the fibrin beta-chain, competes with fibrin fragments for binding to VEcadherin and blocks transmigration. To test effects of peptide Bbeta15-42 in vivo we selected a cardial ischaemia/reperfusion model in rats, where the coronary artery is temporarily occluded and then reopened (mimicking infarction followed by reperfusion treatment through e.g. PTCA in humans). In this model – as in humans – reperfusion causes myocardial inflammation, which leads to myocardial damage cutting down benefits of reperfusion treatment. When peptide Bbeta15-42 was intravenously injected into rats at the time of reperfusion, myocardial inflammation, the resulting infarct sizes and the subsequent scars were significantly reduced ( p < .05). We propose that peptide Bbeta15-42 is a new candidate for reperfusion therapy in humans. Moreover, by preventing the interaction of fibrin fragments with endothelium, this peptide may pave the way to a generation of new anti-inflammatory peptides.
P313 CLONING AND CHARACTERIZATION OF MURINE CD99. Alan R. Schenkel, William A. Muller. Weill Medical College of Cornell University, New York, NY. CD99 is an important molecule for transendothelial migration of human monocytes. We have cloned and begun to characterize mouse CD99 for use in models of inflammatory disease in vivo. Murine CD99 has two long stretches of high homology with human CD99, as well as some structural similarities like a extracellular glycine-rich domain. Murine CD99 is slightly larger than human CD99, approximately 45 kDa vs. 32 kDa apparent molecular weight. This is due to a larger extracellular domain and possibly differences in O-glycosylation. Similar to human CD99, murine CD99 can be detected on hematopoetic cells and tissues by RTPCR and immuno-fluorescence. We have also begun studies using purified rabbit IgG against murine CD99 in two models of inflammation to explore the role of this molecule during leukocyte extravasation in vivo.
P314 CHARACTERIZATION OF THE T CELLS THAT MEDIATE HUMAN MICROVASCULAR REJECTION. Stephen L. Shiao, Jordan S. Pober. Yale University School of Medicine. Graft endothelial cells (EC) are a principal target of acute rejection. We have previously described a model of T cell-mediated EC destruction involving sequential engraftment of immunodeficient SCID/beige C.B-17 mice with vascularized human skin followed by T cells from an allogeneic (to the skin) donor. In adult humans, over half of the circulating alloreactive T cells are memory cells. In our model, depletion of memory T cells from
Poster Abstracts / Cardiovascular Pathology 13 (2004) S80–S138
P307
P309
COMPOSITION AND REGULATION OF THE PECAM-CONTAINING RECYCLING MEMBRANE COMPARTMENT IN ENDOTHELIAL CELLS. Zahra Mamdouh, Xia Chen, William A. Muller. Weill Medical College of Cornell University, Department of Pathology Room C320, 1300 York Avenue. New York NY 10021.
REGULATION AND IMPLICATION IN MIGRATION OF MEMBRANE TYPE-1 MATRIX METALLOPROTEINASE IN HUMAN MONOCYTES. Salomo´n Matı´as-Roma´n, Beatriz G. Ga´lvez, Laura Genı´s, Alicia G. Arroyo. Centro de Investigaciones Biolo´gicas, Spain.
PECAM-1, a glycoprotein expressed on most leukocytes and on endothelial cells (EC) at the cell border, is a key molecule controlling diapedesis. We have recently shown that PECAM-1 enters an intracellular membrane compartment connected to the surface of human umbilical vein endothelial cells (HUVEC). Trafficking of PECAM-1 between this compartment and EC surface occurs evenly along the junction in resting cells. However, during diapedesis, recycling PECAM-1 is targeted preferentially to the zone of the junction surrounding the migrating monocyte. We also found that this compartment is novel, distinct from the endocytic recycling compartment and from caveolae. Furthermore, electron microscopy of postcapillary venules recovered from mice intravenously injected with anti-mouse PECAM-1 showed intracellular structures containing PECAM-1 similar to those we previously described in HUVEC. We investigated whether other junctional molecules were also present in the PECAM-containing membrane compartment. CD99 is in these structures while VE-cadherin is not. We also show that treatment of endothelial cells with demecolcine, a drug that blocks microtubule polymerization, alters the constitutive and targeted recycling of PECAM-1. This treatment also selectively blocks monocyte transendothelial migration by 80% without interfering with their ability to adhere to and crawl on the endothelial cell surface. This implies a role for microtubules in maintaining the structure or the function of this PECAM-containing compartment.
Recruitment of monocytes to tissues is a crucial event in the inflammatory response, not yet completely understood. The metalloproteinase MT1MMP is involved in the degradation of extracellular matrix ( ECM ), and in this regard, its action is important for the migration of different cell types. We demonstrate in this work that binding of human monocytes to fibronectin ( FN ), upregulates the expression of MT1-MMP, through the interaction with integrins a4 and a5. An increase in MT1-MMP is also observed after attachment of monocytes to either resting or activated human endothelial cells. In fact, interaction of monocytes with the endothelial ligands ICAM-1 or VCAM-1 also results in an induction of MT1-MMP expression. Moreover, the increase in MT1-MMP expression correlates with an upregulation of its fibrinolytic activity upon binding of monocytes to either FN or endothelial ligands. In addition, MT1-MMP is located at protrusive plasma membrane structures of migratory human monocytes. Finally, a functional role for MT1-MMP is demonstrated by inhibition of monocyte migration on FN with anti-MT1-MMP mAbs. All these data show that MT1-MMP is regulated by the ECM and the endothelium in human monocytes , and it plays an important role during their migration. Grant SAF2002-00068 from Ministerio de Ciencia y Tecnologı´a (Spain)
supported by NIH grant HL46849 to WAM P310
P308 DISSECTION OF THE ROLES OF ENDOTHELIAL ADHESION MOLECULES AND CHEMOKINES IN RECRUITMENT OF CD4+ T CELLS UNDER CONDITIONS OF PHYSIOLOGICAL FLOW. Thomas Manes, Jordan S. Pober, Martin S. Kluger. Yale University School of Medicine. TNF-treated human umbilical vein endothelial cell ( HUVEC ) monolayers overlaid with the chemokine SDF-1 are able to bind and stimulate the transendothelial migration ( TEM ) of CD4+T cells under conditions of physiologically relevant flow (1 dyne/cm2). Antibody blocking experiments and genetic deletion of specific TNF-induced adhesion molecules, such as E-selectin, ICAM-1, and VCAM-1, have identified roles for these proteins in T cell recruitment when expressed on inflamed endothelium, but it is not known if adhesion molecule expression is sufficient to account for the effects of TNF. To address this question, we prepared retrovirally transduced HUVEC that constitutively (in the absence of cytokine) express E-selectin, ICAM-1, and VCAM-1, either in pairwise combinations or in triple combination. We find that triple adhesion molecule expressing cells are able to support T cell TEM equally as well as TNF-treated EC. Double expressers performed less well and no individual adhesion molecule appeared to be indispensable, although favoring of specific T cell subsets may occur. In the absence of SDF-1, neither TNF-treated nor adhesion molecule-transduced HUVEC were able to stimulate TEM. These observations suggest that, in the presence of chemokine, adhesion molecule expression can largely account for the pro-inflammatory effects of TNF on T cell recruitment by EC and that there is overlap in the functions of Eselectin, ICAM-1, and VCAM-1 expressed on EC.
ACTIVATION OF CALCIUM-INDEPENDENT PHOSPHOLIPASE A2 IN TRYPTASE-STIMULATED HUMAN BLADDER ENDOTHELIAL CELLS. Maureen Meyer, Pamela Kell, Alice Rickard, Jane McHowat. Saint Louis University, Saint Louis, USA. Interstitial cystitis is an inflammatory bladder condition with unknown etiology, although a pathophysiologic role for mast cells has been implicated. We hypothesized that mast cell tryptase may contribute to the exacerbation of inflammation by activating the protease-activated receptor-2 (PAR-2) on human bladder microvascular endothelial cells (HBMEC), resulting in activation of phospholipase A2 (PLA2) and increased production of inflammatory phospholipid metabolites. Incubation of confluent monolayers of HBMEC with tryptase (0.2 to 200 ng/ml for intervals up to 60 mins) resulted in a dose- and time-dependent increase in platelet-activating factor (PAF) production and release of arachidonic acid, prostacyclin and prostaglandin E2. Pretreatment of HBMEC with the calcium-independent PLA2 (iPLA2) inhibitor bromoenol lactone ( BEL, 5 microM, 10 mins) resulted in complete inhibition of tryptase-stimulated production of phospholipid metabolites, suggesting that tryptase stimulates increased iPLA2 activity in HBMEC. Tryptase-simulated increases in PAF production were accompanied by increased expression of P-selectin on the HBMEC cell surface and resulted in a significant increase in adherence of neutrophils to the HBMEC monolayer. Pretreatment of HBMEC with methyl arachidonyl fluorophosphonate (MAFP, 5 microM, 10 mins, a cytosolic PLA2 inhibitor which also inhibits PAF acetylhydrolase activity) resulted in a significant potentiation of tryptase-stimulated PAF production, cell surface expression of P-selectin and neutrophil adherence. These data indicate that tryptase stimulation of HBMEC results in activation of iPLA2, resulting in increased membrane phospholipid hydrolysis and accumulation of several inflammatory mediators. In particular, the increase in cell surface expression of P-selectin and the increased neutrophil adherence to the endothelial cell monolayer following tryptase incubation
Poster Abstracts / Cardiovascular Pathology 13 (2004) S80–S138 suggests that this protease may play an important role in propagation of the inflammatory response. National Institute of Diabetes and Digestive and Kidney Diseases DK066119
P311 TOLL-LIKE RECEPTOR 2 ACTIVATION OF MONOCYTES DECREASES ADHESION TO ENDOTHELIAL-LIKE CELLS UNDER FLOW CONDITIONS. Manon M Oude Nijhuis, Laurien H Ulfman, Arjan H Schoneveld, Dominique PV de Kleijn, Nienke I Sluis, Gerard Pasterkamp. Experimental Cardiology Laboratory, University Medical Center Utrecht, Utrecht, Netherlands, Department of Pulmonary Diseases, University Medical Center Utrecht, Utrecht, Netherlands. Background: Atherosclerosis is associated with an intense inflammatory response. During this response mediators like chemokines and cytokines are able to recruit leukocytes and macrophages to the site of injury. Previously, we demonstrated that peptidoglycan ( PGN ), a Toll-like receptor 2 ( TLR-2 ) ligand, is present in unstable atherosclerotic lesions hiding inflammatory cells. The presence of peptidoglycan was mainly observed in macrophage-rich regions. In addition, we demonstrated a significant relation between IgM antibody levels against this TLR-2 ligand and carotid intima-media thickness. In the present study, we determined the effect of TLR-2 ligands on monocyte adhesion to endothelial-like cell layers under flow conditions. Methods: The TLR-2 ligands used are purified Staphylococcus aureus PGN and synthetic Pam3Cys. On monocytes, activated by these TLR-2 ligands, the expression of adhesion molecules CD18, CD11b and CD62L was measured by flow cytometry. Furthermore, an in vitro flow chamber model was used to study the interaction between PGN- and Pam3Cys- activated monocytes to endothelial-like cells (Chinese Hamster Ovary cells (CHO cells) expressing E-selectin, and L-cells expressing E-selectin and ICAM-1). The role of L-selectin on adhesion of the monocytes to the endothelial-like cells was evaluated by the use of a blocking antibody ( Dreg56 ). Results: Higher levels of the monocyte receptors CD18 and CD11b were expressed on monocytes activated with the TLR-2 ligands compared to control monocytes (CD18; 137.0+/-22.4 versus 79.9+/ 11.0, CD11b; 221.2+/ 57.8 versus 132.1+/ 21.9 for Pam3Cys and CD18; 126.9+/ 22.7 versus 91.4+/ 18.2, CD11b; 185.5+/ 32.5 versus 94.2+/ 16.7 for PGN), while L-selectin (CD62L) was shed from the surface after monocyte activation (10.3+/ 2.8 versus 38.3+/ 6.8 for Pam3Cys and 23.8+/ 10.4 versus 47.2+/ 5.3 for PGN). TLR2 activation resulted in a significantly reduced number of adherent monocytes to both the CHO cells and the Lcells compared to nonactivated control monocytes. This suggests that Lselectin shedding results in a decrease in monocyte tethering to Eselectinexpressing endothelial cells. Indeed, in the presence of an Lselectin blocking antibody adherence of control monocytes dropped to values similar to monocyte adhesion after TLR2 activation. Conclusion: These results show that monocytes increase the expression of adhesion receptors on their surface and shed Lselectin upon TLR2 activation. Surprisingly, however, the adhesion of monocytes to endotheliallike cells is decreased after TLR2 activation probably due to the Lselectin shedding. Future studies will evaluate the role of CD11b, CD18 on TLR2 stimulated monocytes in the shear resistance to endothelial cells. This might reveal the mechanism by which peptidoglycan enters the atherosclerotic plaque.
S115
P312 THE FIBRIN-DERIVED PEPTIDE BB15-42 REDUCES MYOCARDIAL REPERFUSION INJURY. Peter Petzelbauer, Peter Friedl, Marion Gro¨ger, Paula Zacharowski, Klaus Wolff, Kai Zacharowski. Department of Dermatology, Division of General Dermatology, University of Vienna, Medical School, A-1090 Vienna, Austria, Department of Dermatology, Division of General Dermatology, University of Vienna, Medical School, A-1090 Vienna, Austria, Molecular Cardioprotection and Inflammation Group, Department of Anaesthesiology, University Hospital of Du¨sseldorf, D-40225, Du¨sseldorf, Germany. VE-cadherin is typically expressed within junctional regions of neighbouring endothelial cells, where it forms homotypic adhesion complexes. Recently the N-terminal sequence of the fibrin beta-chain was identified as a novel binding partner for VE-cadherin. Unexpectedly, by binding to endothelial VE-cadherin, fibrin fragments induce transmigration of T cells, monocytes and neutrophils across monolayers of endothelial cells in culture. Peptide Bbeta15-42, which matches the N-terminal sequence of the fibrin beta-chain, competes with fibrin fragments for binding to VEcadherin and blocks transmigration. To test effects of peptide Bbeta15-42 in vivo we selected a cardial ischaemia/reperfusion model in rats, where the coronary artery is temporarily occluded and then reopened (mimicking infarction followed by reperfusion treatment through e.g. PTCA in humans). In this model – as in humans – reperfusion causes myocardial inflammation, which leads to myocardial damage cutting down benefits of reperfusion treatment. When peptide Bbeta15-42 was intravenously injected into rats at the time of reperfusion, myocardial inflammation, the resulting infarct sizes and the subsequent scars were significantly reduced ( p < .05). We propose that peptide Bbeta15-42 is a new candidate for reperfusion therapy in humans. Moreover, by preventing the interaction of fibrin fragments with endothelium, this peptide may pave the way to a generation of new anti-inflammatory peptides.
P313 CLONING AND CHARACTERIZATION OF MURINE CD99. Alan R. Schenkel, William A. Muller. Weill Medical College of Cornell University, New York, NY. CD99 is an important molecule for transendothelial migration of human monocytes. We have cloned and begun to characterize mouse CD99 for use in models of inflammatory disease in vivo. Murine CD99 has two long stretches of high homology with human CD99, as well as some structural similarities like a extracellular glycine-rich domain. Murine CD99 is slightly larger than human CD99, approximately 45 kDa vs. 32 kDa apparent molecular weight. This is due to a larger extracellular domain and possibly differences in O-glycosylation. Similar to human CD99, murine CD99 can be detected on hematopoetic cells and tissues by RTPCR and immuno-fluorescence. We have also begun studies using purified rabbit IgG against murine CD99 in two models of inflammation to explore the role of this molecule during leukocyte extravasation in vivo.
P314 CHARACTERIZATION OF THE T CELLS THAT MEDIATE HUMAN MICROVASCULAR REJECTION. Stephen L. Shiao, Jordan S. Pober. Yale University School of Medicine. Graft endothelial cells (EC) are a principal target of acute rejection. We have previously described a model of T cell-mediated EC destruction involving sequential engraftment of immunodeficient SCID/beige C.B-17 mice with vascularized human skin followed by T cells from an allogeneic (to the skin) donor. In adult humans, over half of the circulating alloreactive T cells are memory cells. In our model, depletion of memory T cells from