Modulation of endothelial cell pro-inflammatory phenotype by polymorphonuclear leukocytes (PMN): role of platelet–endothelial cell adhesion molecule-1 (PECAM-1)

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14th IVBM Abstracts

Results: Inhibition of HO-1 by ZnPP prevented DAF upregulation by TNFα/IFNγ and VEGF. Treatment of human EC with CoPP significantly increased HO-1 expression after 6 h and induced DAF surface expression, maximal after 48 h (p < 0.05). This reflected an increase in steady-state DAF mRNA, detectable at 8 h and maximal after 24 h. Adenoviral-mediated over-expression of HO-1 resulted in increased DAF expression (p < 0.05) and protection against C3 deposition and complement-mediated lysis, which was reversed by a DAF inhibitory mAb. Treatment of HUVEC with bilirubin increased DAF mRNA and protein expression and also protected EC against complement activation. Likewise, both CO and iron chelation increased DAF expression (p < 0.05). Analysis of aortic EC from HO-1-deficient and wild-type mice revealed a 60% reduction in DAF expression in HO-1-/-EC and increased sensitivity to complement activation. Conclusions: Induction of HO-1 in EC induces DAF surface expression and protects against complement-mediated injury. The products of HO-1, bilirubin and CO, and the chelation of iron reproduced the effects of HO-1. We suggest that protection against complement-mediated injury is an additional important downstream cytoprotective effect of HO-1 in the vascular endothelium.

microscopy. The obtained results indicate that migration of PMN across IL-1b-stimulated HUVEC and RHP cells leads to the increase in PECAM-1 tyrosine phosphorylation, recruitment of SHP-2 phosphatase and a subsequent decrease in IL-1b-induced degradation of IkB-a. These changes were accompanied by the decrease in NFkB nuclear levels in IL-1b-stimulated HUVEC and RHP. The above effects were absent in PITC cells lacking functional cytoplasmic PECAM-1 domain and EC obtained from PECAM-1-/-mice. In parallel in vivo experiments we assessed the role of PECAM-1 on the general outcome of systemic inflammation. A further increase in the heart and lung tissue NFkB levels was found in septic PECAM-1-/-mice in comparison to their WTcounterparts. This was accompanied by the increase in circulating levels of NFkB-dependent markers of endothelial cell injury (E-selectin/ICAM-1) and impaired survival of septic PECAM-1-/-mice. Taken together these results indicate that engagement of endothelial cell PECAM-1 by migrating PMN is a pivotal event in regulation of a negative feedback inhibition of NFkB and pro-inflammatory phenotype in vascular endothelium during systemic inflammation (HSFO-NA5580). doi:10.1016/j.vph.2006.08.057

doi:10.1016/j.vph.2006.08.056

A1.04 A1.03 Modulation of endothelial cell pro-inflammatory phenotype by polymorphonuclear leukocytes (PMN): Role of platelet–endothelial cell adhesion molecule-1 (PECAM-1) Gediminas Cepinskas1, Osamu Handa2

Platelet binding to monocytes promote monocyte adhesion and transendothelial migration. Do platelets migrate along? Janine M. van Gils1, Paula A. da CostaMartins1, Anita Mol1, Peter L. Hordijk1, Jaap Jan Zwaginga2 1 2

1 2

Lawson Health Research Institute, London, Canada Kyoto Prefectural University of Medicine, Kyoto, Japan

Our recent findings indicate that during systemic inflammation PMN migrating across vascular endothelium can down-regulate inducible nuclear factor kappa B (NFkB), a key transcription factor capable of modulating inflammatory response, through the novel mechanism involving PECAM-1. In this study we used both, in vitro and in vivo approaches to assess the molecular mechanisms of PECAM-1 signaling associated to the above phenomenon. We employed human umbilical vein endothelial cells (HUVEC) and EC-like mesothelioma cells RHP and PITC, transfected with a full or chimeric (containing ICAM-1 cytoplasmic domain) PECAM-1, respectively to address the specific questions. To this end EC were stimulated with IL-1b (1 ng/ml) and interacted with PMN in PMN transendothelial migration assay. Subsequently, EC PECAM-1 was immunoprecipitated and assessed (Western blot) for tyrosine phosphorylation, recruitment of SHP-2 phosphatase along with the cytoplasmic levels of NFkB inhibitory protein, IkB. In parallel, NFkB nuclear levels were assessed by EMSA and confocal

Sanquin Research, Amsterdam, The Netherlands Leiden University Medical Center, Leiden, The Netherlands

Monocyte adhesion to and transmigration across the endothelium are essential steps in atherogenesis. We previously reported that the adhesive capacity of monocytes is increased when platelets bind to the monocyte surface and form platelet– monocyte complexes (PMC) via P-selectin on the platelets and PSGL-1 on the monocytes. To better understand the effect of platelet binding on the adhesive capacity of monocytes, we now studied the changes in integrin functionality in PMC. By flowcytometry and simultaneous blockade of interactions with MoAbs, we could show that binding of P-selectin to PSGL-1 on monocytes increased expression and activation of α4β1- and αMβ2-integrins. Functionality was confirmed by increased PMC adhesion to integrin ligands ICAM-1, and VCAM-1, and fibronectin. Additionally, platelet binding also enhanced monocyte chemotaxis. However, the level of PMC in the migrated fraction seemed reduced. Therefore, the fate of platelets (within the PMC) during monocyte transendothelial migration was further investigated. PMC were seeded on endothelial cells that were cultured on top of a fibrin gel, and the position of platelets and monocytes after migration was analyzed by confocal laser