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EFFECT OF ICAM-1 POLYMORPHISM ON ENDOTHELIAL CELLS IN VITRO
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Poster Abstracts / Cardiovascular Pathology 13 (2004) S80–S138
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IL-11 PROTECTS HUMAN MICROVASCULAR ENDOTHELIUM FROM ALLOINJURY IN VIVO BY INDUCTION OF SURVIVIN EXPRESSION. Nancy C. Kirkiles-Smith, Keyvan Mahboubi, Janet Plescia, Jennifer M. McNiff, James Karras, Jeffrey S. Schechner, Dario C. Altieri, Jordan S. Pober. Yale University School of Medicine, New Haven CT, Yale University School of Medicine, New Haven CT, University of Massachusetts School of Medicine, Worcester MA, ISIS Pharmaceuticals Carlsbad, CA.
ANGIOPOIETIN-1 AND -2 MEDIATE PROINFLAMMATORY RESPONSES IN HUMAN ENDOTHELIAL CELLS. Caroline Lemieux, Ricardo Maliba, Yahye Merhi, Martin G. Sirois. Montreal Heart Institute & University of Montreal, Montreal, Quebec, Canada.
Microvascular endothelial cells ( EC ) in vascularized allografts are primary targets of acute allogeneic rejection responses. Protection of graft EC by induction of cytoprotective proteins may decrease immune-mediated injury and prolong graft survival. IL-11, a pleiotropic cytokine, has previously been shown to have anti-inflammatory and immunomodulatory activities in vivo. Studies from our laboratory using cultured human EC have additionally demonstrated cytoprotective action in vitro. In the current study we used a huPBL-SCID/human skin allograft model that allows for examination of human T cell interactions with human dermal microvessels in an immunodeficient mouse host. When C.B-17 SCID/beige mice bearing human skin grafts are injected intraperitoneally with human PBMC allogeneic to the donor skin, infiltrating T cells destroy human microvessels by day 21. Intradermal injection of human IL-11 (500 ng/day) reduces graft microvessel loss without reducing the extent of T cell infiltration assessed on day 15. IL-11 treatment has no effect on T cell activation markers (CD25 and CD69), effector molecule expression (FasL, Granzyme B and perforin), cytokine expression ( IFN-g or IL-5 ), or on endothelial ICAM-1 expression assessed by quantitative real-time RT-PCR. IL-11 up-regulates the expression of survivin, a cytoprotective protein, in graft keratinocytes and EC. Topical application of survivin antisense oligonucleotide down-regulates survivin mRNA and protein expression in both cell types and largely abrogates the protective effect of IL-11. The effects of antisense are specific for survivin and did not reduce levels of SOCS-3. We conclude that in this human transplant model, IL-11 exerts a cytoprotective rather than antiinflammatory or immunomodulatory effect and the cytoprotection is mediated through induction of survivin.
P302 EFFECT OF ICAM-1 POLYMORPHISM ON ENDOTHELIAL CELLS IN VITRO. Charlotte Lawson, Angela Holder, Marlene Rose. Imperial College, London, UK. Intercellular adhesion molecule ( ICAM )-1 is critical for firm arrest and transendothelial migration of leukocytes. Two single base pair polymorphisms have been described for ICAM-1, located in exons 4 and 6, modifying codons 241 (G/R; Mac-1 binding domain) and 469 ( E/K; aggregation domain). Associations of these polymorphic residues with diseases have been identified but the functional significance has not been addressed. Here cDNA constructs of each ICAM-1 polymorphic variant have been made and transfected into Cos 7 cells. Using a panel of MAbs (6.5B5, 8.4A6, 7.5C2, RR 1/ 1, MEM-111, MEM-112) directed against different epitopes of ICAM-1 extracellular domain there was increased expression of ICAM-1 of GE genotype (GE p > .05 compared to expression of GK or RE all MAbs). A murine cardiac endothelial cell line (Am.J.Physiol.Cell.Physiol 282:C67 2002) was infected with adenoviruses encoding each ICAM-1 polymorphic variant. Adhesion of fluorescently labelled K562 cells transfected with a constitutively active form of LFA-1 (Mol. Biol. of the Cell 8:719 1997) was increased to cells infected with Ad5 encoding ICAM-1 of GE genotype. Activation of extracellular regulated kinase (ERK)-1 and-2 was also increased after co-culture of cells expressing GE variant of ICAM-1 with LFA-1-transfected K562. These findings may be of functional significance in the disease states where associations with ICAM-1 polymorphisms have been described. British Heart Foundation
Angiogenesis is a multistep process characterized by the formation of new blood vessels from the preexisting vasculature. Maturation and stabilization of the vascular wall are critically regulated by angiopoietin-1 (Ang-1), upon the activation of Tie2 receptor on endothelial cells. On the other hand, Ang2, which has an equivalent binding affinity to Tie2, was initially reported to be an endogenous antagonist of this receptor. However, recent findings suggest that Ang-2 may, under certain circumstances, act as a Tie2 agonist. By inducing vessel destabilization, Ang-2 activity could potentiate the angiogenic activity of the Vascular Endothelial Growth Factor (VEGF). Since our laboratory has shown that the inflammatory effect of VEGF promotes the translocation of P-selectin, and mediates adhesion of neutrophils to the endothelial cell surface, we then wanted to assess if Ang-1 and/or Ang-2 can participate to these processes. Endothelial P-selectin translocation and neutrophil adhesion to confluent human umbilical vein endothelial cells (HUVEC) were assessed by cell surface ELISA and cell surface adhesion under static conditions, respectively. Tie2 phosphorylaion in HUVEC was evaluated by Western blot analysis. Treatment of HUVEC with Ang-1 and Ang-2 (10-12 M-10-8 M) induced a rapid and transient translocation of P-selectin, the maximal effect being achieved at 10-9 M (273% and 253% induction respectively) within 7.5 minutes. The same experimental conditions led to the adhesion of neutrophils to HUVEC. Maximal effects initiated by Ang-1 and Ang-2 were also achieved at 10-9 M (350% and 340% increase, respectively) within 7.5 minutes of treatment. Western blot analysis revealed that both Ang-1 and Ang-2 (10-9 M) increased Tie2 phosphorylation by 504% and 60% respectively. We then assessed if the combination of Ang-1 and Ang-2 (10-9 M) would affect their corresponding biological activities. No significant increase was observed with respect to P-selectin translocation. Surprisingly, while the combination of both angiopoietins reduced Tie2 phosphorylation to 386% with respect to Ang-1, it elicited an additive effect on neutrophil adhesion to HUVEC. In summary, our data reveal that Ang-2 is able to phosphorylate Tie2 and has an equipotent agonist activity as compared to Ang-1 on P-selectin translocation and neutrophil adhesion to HUVEC. Furthermore, the combination of Ang-1 and Ang-2 might induce the expression and/or the translocation of other mediators thereby contributing to enhance the adhesion of neutrophils to endothelial cells. Consequently, these findings suggest a potential role for both angiopoietins in the induction of acute inflammatory responses. This study was supported by CIHR, HSFQ and FRICM grants.
P304 NEUTROPHILS UTILIZE CD99 DURING TRANSENDOTHELIAL MIGRATION. Olivia Lou, William A. Muller. Department of Pathology, Weill Medical College of Cornell University, New York, NY 10021. Neutrophils ( PMN ) circulating through the blood arrest and transmigrate across endothelial cells upon encountering inflammatory stimuli. We are using an in vitro model of inflammation to dissect the molecular mechanisms that regulate neutrophil transendothelial migration ( TEM ). PECAM (CD31) is a critical molecule in monocyte and neutrophil TEM, and our laboratory recently found that CD99 has a role in monocyte transmigration. CD99 is a transmembrane glycoprotein expressed on most hematopoietic cell types and concentrated at endothelial cell borders. It has no homology to any protein, and its functions and mechanisms of action remain largely unknown. Neutrophils express much less CD99 than monocytes: by FACS, the mean fluorescence intensity is 5 – 10 times lower, making it unapparent whether
Poster Abstracts / Cardiovascular Pathology 13 (2004) S80–S138
P301
P303
IL-11 PROTECTS HUMAN MICROVASCULAR ENDOTHELIUM FROM ALLOINJURY IN VIVO BY INDUCTION OF SURVIVIN EXPRESSION. Nancy C. Kirkiles-Smith, Keyvan Mahboubi, Janet Plescia, Jennifer M. McNiff, James Karras, Jeffrey S. Schechner, Dario C. Altieri, Jordan S. Pober. Yale University School of Medicine, New Haven CT, Yale University School of Medicine, New Haven CT, University of Massachusetts School of Medicine, Worcester MA, ISIS Pharmaceuticals Carlsbad, CA.
ANGIOPOIETIN-1 AND -2 MEDIATE PROINFLAMMATORY RESPONSES IN HUMAN ENDOTHELIAL CELLS. Caroline Lemieux, Ricardo Maliba, Yahye Merhi, Martin G. Sirois. Montreal Heart Institute & University of Montreal, Montreal, Quebec, Canada.
Microvascular endothelial cells ( EC ) in vascularized allografts are primary targets of acute allogeneic rejection responses. Protection of graft EC by induction of cytoprotective proteins may decrease immune-mediated injury and prolong graft survival. IL-11, a pleiotropic cytokine, has previously been shown to have anti-inflammatory and immunomodulatory activities in vivo. Studies from our laboratory using cultured human EC have additionally demonstrated cytoprotective action in vitro. In the current study we used a huPBL-SCID/human skin allograft model that allows for examination of human T cell interactions with human dermal microvessels in an immunodeficient mouse host. When C.B-17 SCID/beige mice bearing human skin grafts are injected intraperitoneally with human PBMC allogeneic to the donor skin, infiltrating T cells destroy human microvessels by day 21. Intradermal injection of human IL-11 (500 ng/day) reduces graft microvessel loss without reducing the extent of T cell infiltration assessed on day 15. IL-11 treatment has no effect on T cell activation markers (CD25 and CD69), effector molecule expression (FasL, Granzyme B and perforin), cytokine expression ( IFN-g or IL-5 ), or on endothelial ICAM-1 expression assessed by quantitative real-time RT-PCR. IL-11 up-regulates the expression of survivin, a cytoprotective protein, in graft keratinocytes and EC. Topical application of survivin antisense oligonucleotide down-regulates survivin mRNA and protein expression in both cell types and largely abrogates the protective effect of IL-11. The effects of antisense are specific for survivin and did not reduce levels of SOCS-3. We conclude that in this human transplant model, IL-11 exerts a cytoprotective rather than antiinflammatory or immunomodulatory effect and the cytoprotection is mediated through induction of survivin.
P302 EFFECT OF ICAM-1 POLYMORPHISM ON ENDOTHELIAL CELLS IN VITRO. Charlotte Lawson, Angela Holder, Marlene Rose. Imperial College, London, UK. Intercellular adhesion molecule ( ICAM )-1 is critical for firm arrest and transendothelial migration of leukocytes. Two single base pair polymorphisms have been described for ICAM-1, located in exons 4 and 6, modifying codons 241 (G/R; Mac-1 binding domain) and 469 ( E/K; aggregation domain). Associations of these polymorphic residues with diseases have been identified but the functional significance has not been addressed. Here cDNA constructs of each ICAM-1 polymorphic variant have been made and transfected into Cos 7 cells. Using a panel of MAbs (6.5B5, 8.4A6, 7.5C2, RR 1/ 1, MEM-111, MEM-112) directed against different epitopes of ICAM-1 extracellular domain there was increased expression of ICAM-1 of GE genotype (GE p > .05 compared to expression of GK or RE all MAbs). A murine cardiac endothelial cell line (Am.J.Physiol.Cell.Physiol 282:C67 2002) was infected with adenoviruses encoding each ICAM-1 polymorphic variant. Adhesion of fluorescently labelled K562 cells transfected with a constitutively active form of LFA-1 (Mol. Biol. of the Cell 8:719 1997) was increased to cells infected with Ad5 encoding ICAM-1 of GE genotype. Activation of extracellular regulated kinase (ERK)-1 and-2 was also increased after co-culture of cells expressing GE variant of ICAM-1 with LFA-1-transfected K562. These findings may be of functional significance in the disease states where associations with ICAM-1 polymorphisms have been described. British Heart Foundation
Angiogenesis is a multistep process characterized by the formation of new blood vessels from the preexisting vasculature. Maturation and stabilization of the vascular wall are critically regulated by angiopoietin-1 (Ang-1), upon the activation of Tie2 receptor on endothelial cells. On the other hand, Ang2, which has an equivalent binding affinity to Tie2, was initially reported to be an endogenous antagonist of this receptor. However, recent findings suggest that Ang-2 may, under certain circumstances, act as a Tie2 agonist. By inducing vessel destabilization, Ang-2 activity could potentiate the angiogenic activity of the Vascular Endothelial Growth Factor (VEGF). Since our laboratory has shown that the inflammatory effect of VEGF promotes the translocation of P-selectin, and mediates adhesion of neutrophils to the endothelial cell surface, we then wanted to assess if Ang-1 and/or Ang-2 can participate to these processes. Endothelial P-selectin translocation and neutrophil adhesion to confluent human umbilical vein endothelial cells (HUVEC) were assessed by cell surface ELISA and cell surface adhesion under static conditions, respectively. Tie2 phosphorylaion in HUVEC was evaluated by Western blot analysis. Treatment of HUVEC with Ang-1 and Ang-2 (10-12 M-10-8 M) induced a rapid and transient translocation of P-selectin, the maximal effect being achieved at 10-9 M (273% and 253% induction respectively) within 7.5 minutes. The same experimental conditions led to the adhesion of neutrophils to HUVEC. Maximal effects initiated by Ang-1 and Ang-2 were also achieved at 10-9 M (350% and 340% increase, respectively) within 7.5 minutes of treatment. Western blot analysis revealed that both Ang-1 and Ang-2 (10-9 M) increased Tie2 phosphorylation by 504% and 60% respectively. We then assessed if the combination of Ang-1 and Ang-2 (10-9 M) would affect their corresponding biological activities. No significant increase was observed with respect to P-selectin translocation. Surprisingly, while the combination of both angiopoietins reduced Tie2 phosphorylation to 386% with respect to Ang-1, it elicited an additive effect on neutrophil adhesion to HUVEC. In summary, our data reveal that Ang-2 is able to phosphorylate Tie2 and has an equipotent agonist activity as compared to Ang-1 on P-selectin translocation and neutrophil adhesion to HUVEC. Furthermore, the combination of Ang-1 and Ang-2 might induce the expression and/or the translocation of other mediators thereby contributing to enhance the adhesion of neutrophils to endothelial cells. Consequently, these findings suggest a potential role for both angiopoietins in the induction of acute inflammatory responses. This study was supported by CIHR, HSFQ and FRICM grants.
P304 NEUTROPHILS UTILIZE CD99 DURING TRANSENDOTHELIAL MIGRATION. Olivia Lou, William A. Muller. Department of Pathology, Weill Medical College of Cornell University, New York, NY 10021. Neutrophils ( PMN ) circulating through the blood arrest and transmigrate across endothelial cells upon encountering inflammatory stimuli. We are using an in vitro model of inflammation to dissect the molecular mechanisms that regulate neutrophil transendothelial migration ( TEM ). PECAM (CD31) is a critical molecule in monocyte and neutrophil TEM, and our laboratory recently found that CD99 has a role in monocyte transmigration. CD99 is a transmembrane glycoprotein expressed on most hematopoietic cell types and concentrated at endothelial cell borders. It has no homology to any protein, and its functions and mechanisms of action remain largely unknown. Neutrophils express much less CD99 than monocytes: by FACS, the mean fluorescence intensity is 5 – 10 times lower, making it unapparent whether