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Peter J. Newman - One of the best experts on this subject based on the ideXlab platform.
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regulation of endothelial cell barrier function by antibody driven affinity modulation of platelet endothelial cell adhesion molecule 1 pecam 1
Journal of Biological Chemistry, 2014Co-Authors: Jay M Campbell, Peter J. Newman, Cathy Paddock, Panida Lertkiatmongkol, Michael W Mosesson, Ralph M AlbrechtAbstract:Abstract PECAM-1 is a 130-kDa member of the immunoglobulin (Ig) superfamily that is expressed on the surface of platelets and leukocytes, and at the intracellular junctions of confluent endothelial cell monolayers. Previous studies have shown that PECAM-1/PECAM-1 homophilic interactions play a key role in leukocyte transendothelial migration, in allowing PECAM-1 to serve as a mechanosensory complex in endothelial cells, in its ability to confer cytoprotection to proapoptotic stimuli, and in maintaining endothelial cell junctional integrity. To examine the adhesive properties of full-length PECAM-1 in a native lipid environment, we purified it from platelets and assembled it into phospholipid nanodiscs. PECAM-1-containing nanodiscs retained not only their ability to bind homophilically to PECAM-1-expressing cells, but exhibited regulatable adhesive interactions that could be modulated by ligands that bind membrane-proximal Ig Domain 6. This property was exploited to enhance the rate of barrier restoration in endothelial cell monolayers subjected to inflammatory challenge. The finding that the adhesive properties of PECAM-1 are regulatable suggests novel approaches for controlling endothelial cell migration and barrier function in a variety of vascular permeability disorders.
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Lyn and PECAM-1 function as interdependent inhibitors of platelet aggregation.
Blood, 2011Co-Authors: Zhang-yin Ming, Peter J. Newman, Jizhou Xiang, Peter Polewski, Debra K. NewmanAbstract:Inhibition of platelet responsiveness is important to control pathologic thrombus formation. Platelet-endothelial cell adhesion molecule-1 (PECAM-1) and the Src family kinase Lyn inhibit platelet activation by the glycoprotein VI (GPVI) collagen receptor; however, it is not known whether PECAM-1 and Lyn function in the same or different inhibitory pathways. In these studies, we found that, relative to wild-type platelets, platelets derived from PECAM-1-deficient, Lyn-deficient, or PECAM-1/Lyn double-deficient mice were equally hyperresponsive to stimulation with a GPVI-specific agonist, indicating that PECAM-1 and Lyn participate in the same inhibitory pathway. Lyn was required for PECAM-1 tyrosine phosphorylation and subsequent binding of the Src homology 2 domain-containing phosphatase-2, SHP-2. These results support a model in which PECAM-1/SHP-2 complexes, formed in a Lyn-dependent manner, suppress GPVI signaling.
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Inhibition of antigen-receptor signaling by Platelet Endothelial Cell Adhesion Molecule-1 (CD31) requires functional ITIMs, SHP-2, and p56(lck).
Blood, 2001Co-Authors: Debra K. Newman, Christin Hamilton, Peter J. NewmanAbstract:Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1, CD31) is a 130-kd member of the immunoglobulin gene superfamily that is expressed on the surface of platelets, endothelial cells, myeloid cells, and certain lymphocyte subsets. PECAM-1 has recently been shown to contain functional immunoreceptor tyrosine-based inhibitory motifs (ITIMs) within its cytoplasmic domain, and co-ligation of PECAM-1 with the T-cell antigen receptor (TCR) results in tyrosine phosphorylation of PECAM-1, recruitment of Src homology 2 domain-containing protein tyrosine phosphatase-2 (SHP-2), and attenuation of TCR-mediated cellular signaling. To determine the molecular basis of PECAM-1 inhibitory signaling in lymphocytes, the study sought to (1) establish the importance of the PECAM-1 ITIMs for its inhibitory activity, (2) determine the relative importance of SHP-2 versus SHP-1 in mediating the inhibitory effect of PECAM-1, and (3) identify the protein tyrosine kinases required for PECAM-1 tyrosine phosphorylation in T cells. Coligation of wild-type PECAM-1 with the B-cell antigen receptor expressed on chicken DT40 B cells resulted in a marked reduction of calcium mobilization—similar to previous observations in T cells. In contrast, co-ligation of an ITIM-less form of PECAM-1 had no inhibitory effect. Furthermore, wild-type PECAM-1 was unable to attenuate calcium mobilization in SHP2‐deficient DT40 variants despite abundant levels of SHP-1 in these cells. Finally, PECAM-1 failed to become tyrosine phosphorylated in p56lck-deficient Jurkat T cells. Together, these data provide important insights into the molecular requirements for PECAM-1 regulation of antigen receptor signaling. (Blood. 2001; 97:2351-2357)
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pecam 1 cd31 expression modulates bleeding time in vivo
American Journal of Pathology, 2000Co-Authors: Sepi Mahooti, Peter J. Newman, Donnasue Graesser, Sonali Patil, Gordon S Duncan, Tak W Mak, Joseph A MadriAbstract:PECAM-1 is a 130-kd member of the Ig superfamily present on endothelial cells, platelets, polymorphonuclear leukocytes, monocytes, and lymphocytes. Its expression begins early in development and persists through adulthood. PECAM-1 functions as an adhesion and signaling molecule between adjacent endothelial cells and between endothelial cells and circulating blood elements. Antibodies directed against PECAM-1 have been shown to affect angiogenesis, endothelial cell migration, and polymorphonuclear leukocyte transmigration. Furthermore, its dimerization is associated with the modulation of integrin affinity. Antibody inhibition studies suggest that PECAM-1 plays a role in modulating thrombosis; however, recent in vitro aggregation studies performed on platelets harvested from PECAM-1-deficient mice revealed no abnormalities. In this report we demonstrate prolonged in vivo bleeding times in PECAM-1-deficient mice. This abnormality was not corrected when wild-type hematopoietic precursors were engrafted into marrow-ablated PECAM-1-deficient mice. Furthermore, normal bleeding times were observed when marrow-ablated wild-type mice were engrafted with hematopoietic precursors harvested from PECAM-1-deficient mice. These studies are consistent with a role for PECAM-1 in modulating thrombosis in the vasculature, which is potentially mediated by endothelial cell PECAM-1 expression.
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a new role for platelet endothelial cell adhesion molecule 1 cd31 inhibition of tcr mediated signal transduction
Journal of Immunology, 1999Co-Authors: Debra K Newtonnash, Peter J. NewmanAbstract:Platelet-endothelial cell adhesion molecule-1 (PECAM-1) is a 130-kDa transmembrane glycoprotein expressed by endothelial cells, platelets, monocytes, neutrophils, and certain T cell subsets. The PECAM-1 extracellular domain has six Ig-homology domains that share sequence similarity with cellular adhesion molecules. The PECAM-1 cytoplasmic domain contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) that, when appropriately engaged, becomes phosphorylated on tyrosine residues, creating docking sites for nontransmembrane, Src homology 2 domain-bearing protein tyrosine phosphatase (SHP)-1 and SHP-2. The purpose of the present study was to determine whether PECAM-1 inhibits protein tyrosine kinase (PTK)-dependent signal transduction mediated by the immunoreceptor tyrosine-based activation motif-containing TCR. Jurkat cells, which coexpress PECAM-1 and the TCR/CD3 complex, were INDO-1AM-labeled and then incubated with anti-CD3e mAbs, anti-PECAM-1 mAbs, or both, and goat anti-mouse IgG was used to cross-link surface-bound mAbs. Calcium mobilization induced by CD3 cross-linking was found to be attenuated by coligation of PECAM-1 in a dose-dependent manner. PECAM-1-mediated inhibition of TCR signaling was attributable, at least in part, to inhibition of release of calcium from intracellular stores. These data provide evidence that PECAM-1 can dampen signals transduced by ITAM-containing receptors and support inclusion of PECAM-1 within the family of ITIM-containing inhibitors of PTK-dependent signal transduction.
Steven M Albelda - One of the best experts on this subject based on the ideXlab platform.
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streptavidin facilitates internalization and pulmonary targeting of an anti endothelial cell antibody platelet endothelial cell adhesion molecule 1 a strategy for vascular immunotargeting of drugs
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: Vladimir R Muzykantov, Melpo Christofidousolomidou, Irina V Balyasnikova, David W Harshaw, Linda Schultz, Aron B Fisher, Steven M AlbeldaAbstract:Conjugation of drugs with antibodies to surface endothelial antigens is a potential strategy for drug delivery to endothelium. We studied antibodies to platelet-endothelial adhesion molecule 1 (PECAM-1, a stably expressed endothelial antigen) as carriers for vascular immunotargeting. Although 125I-labeled anti-PECAM bound to endothelial cells in culture, the antibody was poorly internalized by the cells and accumulated poorly after intravenous administration in mice and rats. However, conjugation of biotinylated anti-PECAM (b-anti-PECAM) with streptavidin (SA) markedly stimulated uptake and internalization of anti-PECAM by endothelial cells and by cells expressing PECAM. In addition, conjugation with streptavidin markedly stimulated uptake of 125I-labeled b-anti-PECAM in perfused rat lungs and in the lungs of intact animals after either intravenous or intraarterial injection. The antioxidant enzyme catalase conjugated with b-anti-PECAM/SA bound to endothelial cells in culture, entered the cells, escaped intracellular degradation, and protected the cells against H2O2-induced injury. Anti-PECAM/SA/125I-catalase accumulated in the lungs after intravenous injection or in the perfused rat lungs and protected these lungs against H2O2-induced injury. Thus, modification of a poor carrier antibody with biotin and SA provides an approach for facilitation of antibody-mediated drug targeting. Anti-PECAM/SA is a promising candidate for vascular immunotargeting of bioactive drugs.
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an anti platelet endothelial cell adhesion molecule 1 antibody inhibits leukocyte extravasation from mesenteric microvessels in vivo by blocking the passage through the basement membrane
Journal of Experimental Medicine, 1996Co-Authors: M W Wakelin, M J Sanz, A Dewar, Steven M Albelda, Simon W Larkin, N Boughtonsmith, T J Williams, Sussan NoursharghAbstract:Platelet-endothelial cell adhesion molecule-1 (PECAM-1, CD31) plays an active role in the process of leukocyte migration through cultured endothelial cells in vitro and anti-PECAM-1 antibodies (Abs) inhibit accumulation of leukocytes into sites of inflammation in vivo. Despite the latter, it is still not clear at which stage of leukocyte emigration in vivo PECAM-1 is involved. To address this point directly, we studied the effect of an anti-PECAM-1 Ab, recognizing rat PECAM-1, on leukocyte responses within rat mesenteric microvessels using intravital microscopy. In mesenteric preparations activated by interleukin (IL)-1 beta, the anti-PECAM-1 Ab had no significant effect on the rolling or adhesion of leukocytes, but inhibited their migration into the surrounding extravascular tissue in a dose-dependent manner. Although in some vessel segments these leukocytes had come to a halt within the vascular lumen, often the leukocytes appeared to be trapped within the vessel wall. Analysis of these sections by electron microscopy revealed that the leukocytes had passed through endothelial cell junctions but not the basement membrane. In contrast to the effect of the Ab in mesenteric preparations treated with IL-1 beta, leukocyte extravasation induced by topical or intraperitoneal administration of the chemotactic peptide formyl-methionyl-leucyl-phenylalanine was not inhibited by the anti-PECAM-1 Ab. These results directly demonstrate a role for PECAM-1 in leukocyte extravasation in vivo and indicate that this involvement is selective for leukocyte extravasation elicited by certain inflammatory mediators. Further, our findings provide the first in vivo indication that PECAM-1 may have an important role in triggering the passage of leukocytes through the perivascular basement membrane.
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individually distinct ig homology domains in pecam 1 regulate homophilic binding and modulate receptor affinity
Journal of Biological Chemistry, 1996Co-Authors: Qihong Sun, Steven M Albelda, Peter J. Newman, Horace M Delisser, Mark M Zukowski, Cathy PaddockAbstract:Abstract PECAM-1 (CD31) is a 130-kDa member of the immunoglobulin (Ig) gene superfamily that is constitutively expressed at high concentration at endothelial cell intercellular junctions and at moderate density on the surface of circulating leukocytes and platelets. Recent in vitro and in vivo studies have shown that PECAM-1 plays a central role in mediating the extravasation of leukocytes from the vessel wall in response to inflammatory mediators. To study the binding characteristics of PECAM-1, phospholipid vesicles were prepared and examined by flow cytometry and immunofluorescence microscopy for their ability to associate with each other and with cells. Proteoliposomes containing high concentrations of PECAM-1 interacted homophilically with each other, forming large self-aggregates. PECAM-1 proteoliposomes, as well as soluble bivalent PECAM-1 in the form of a PECAM-1/IgG immunoadhesin, associated homophilically with cells expressing human, but not murine, PECAM-1. This binding could be completely inhibited by monoclonal antibody Fab fragments specific for Ig homology Domain 1 or Domains 1 + 2. Binding studies using cells expressing human PECAM-1 deletion mutants and murine/human chimeras confirmed that both Ig Domains 1 and 2 were both necessary and sufficient for homophilic binding. In contrast, engagement of membrane-proximal Domain 6 with monoclonal antibody Fab fragments had the opposite effect and augmented the binding of PECAM-1 proteoliposomes to cells. Thus, PECAM-1, like certain integrins, appears to be capable of antibody-induced conformational changes that alter affinity for its ligand. Similar changes induced by physiologic stimuli could be important in regulating the function of PECAM-1 in vascular cells.
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platelet endothelial cell adhesion molecule 1 cd31 mediated cellular aggregation involves cell surface glycosaminoglycans
Journal of Biological Chemistry, 1993Co-Authors: Horace M Delisser, William A Muller, Steven M Albelda, Peter J. Newman, Clayton A Buck, Horng Chin YanAbstract:Platelet/endothelial cell adhesion molecule-1 (PECAM-1, CD31) is a 130-kDa integral membrane glycoprotein expressed on endothelial cells, platelets, and leukocytes. Experiments analyzing the aggregation of mouse L-cells stably transfected with full-length PECAM-1 cDNA have demonstrated that PECAM-1 is capable of mediating calcium-dependent heterophilic aggregation. In this report the ligand interactions involved in the aggregation process were studied. This aggregation was inhibited by heparin and chondroitin sulfate, but not by other glycosaminoglycans. Enzymatic removal of cell surface glycosaminoglycans confirmed a PECAM-1-glycosaminoglycan interaction and suggested that this interaction involved glycosaminoglycans on adjacent cells. PECAM-1 contains a glycosaminoglycan consensus binding sequence in the second immunoglobulin-like domain of the molecule's extracellular domain. A comparable region in the related adhesion protein N-CAM has been shown to mediate the adhesive properties of N-CAM. Cells expressing mutant PECAM-1 protein missing the second domain failed to aggregate. Synthetic peptides mimicking the consensus glycosaminoglycan binding sequence, L-K-R-E-K-N, inhibited aggregation. These results demonstrate that PECAM-1-mediated aggregation is dependent on the binding of PECAM-1 to specific glycosaminoglycans on adjacent cells via a glycosaminoglycan consensus binding sequence in the second immunoglobulin-like homology domain.
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a heterophilic adhesion mechanism for platelet endothelial cell adhesion molecule 1 cd31
Journal of Experimental Medicine, 1992Co-Authors: William A Mulle, Miriam E Erma, Pete J Newma, Horace M Delisse, Steven M AlbeldaAbstract:The molecular nature of cell adhesion mediated by platelet/endothelial cell adhesion molecule 1 (PECAM-1; CD31) was examined using stably transfected L cells in a PECAM-dependent aggregation assay. This adhesion was temperature sensitive and divalent cation dependent, with Mg2+ supporting aggregation to a greater degree than Ca2+. PECAM-dependent aggregation was heterophilic: PECAM-1 transfectants bound as readily to control-transfected L cells as to other PECAM-1 transfectants, demonstrating that a molecule endogenously expressed on the L cells serves as the ligand for PECAM in this system and presumably substitutes for the natural human ligand.
William A Muller - One of the best experts on this subject based on the ideXlab platform.
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platelet endothelial cell adhesion molecule deficiency or blockade significantly reduces leukocyte emigration in a majority of mouse strains
Journal of Immunology, 2004Co-Authors: Alan R Schenkel, Tina W Chew, William A MullerAbstract:PECAM is a molecule used specifically during the diapedesis step when neutrophils and monocytes leave the blood compartment. Anti-PECAM reagents, such as Abs and soluble fusion proteins, block diapedesis both in vivo and in vitro. However, the PECAM knockout mouse in C57BL/6 strain has no serious defects in most models of inflammation. We show in this study that the same PECAM knockout backcrossed into the FVB/n strain clearly has reduced leukocyte emigration in two models of inflammation. Furthermore, we show that anti-PECAM reagents can block leukocyte emigration in several other wild-type strains of mice like FVB/n, SJL, and the outbred strain Swiss Webster. This clearly shows that the C57BL/6 strain is uniquely able to compensate for the loss of PECAM function. Murine models of inflammatory disease that have been studied using C57BL/6 mice should be re-evaluated using FVB/n or other mouse strains to determine whether PECAM plays a role in those models.
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neutrophil platelet endothelial cell adhesion molecule 1 participates in neutrophil recruitment at inflammatory sites and is down regulated after leukocyte extravasation
Journal of Immunology, 1997Co-Authors: Melpo Christofidousolomidou, William A Muller, Marian T Nakada, John L Williams, Horace M DelisserAbstract:Platelet endothelial cell adhesion molecule-1 (PECAM-1), a member of the Ig superfamily, is found on endothelial cells and neutrophils, and has been shown to be involved in the migration of leukocytes across the endothelium. Although studies have supported a role for endothelial PECAM-1 in this process, the participation of neutrophil PECAM-1 in vivo has not been unambiguously demonstrated. Therefore, to examine the involvement of neutrophil PECAM-1 in leukocyte recruitment, we studied the effect of a blocking Ab against murine PECAM-1 on neutrophil recruitment in an established model of murine peritonitis and in a murine model for studying leukocyte-endothelial interactions involving the human vasculature. These studies not only confirmed that neutrophil PECAM-1 is important in the accumulation of neutrophils at inflammatory sites, but that extravasated neutrophils displayed decreased surface expression of PECAM-1. In vitro, the surface expression of murine neutrophil PECAM-1 was not decreased significantly by inflammatory mediators, but was reduced after transendothelial migration. These studies, consistent with previous in vitro observations, confirm that neutrophil PECAM-1, as well as endothelial PECAM-1, is involved in the recruitment of neutrophils into inflammatory sites in vivo, and suggest that the expression of neutrophil PECAM-1 is down-regulated after extravasation into inflamed tissues possibly as a result of engagement of its ligand.
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roles of platelet endothelial cell adhesion molecule 1 pecam 1 cd31 in natural killer cell transendothelial migration and beta 2 integrin activation
Journal of Immunology, 1996Co-Authors: Miriam E Berman, Yong Xie, William A MullerAbstract:Platelet/endothelial cell adhesion molecule-1 (PECAM-1, CD31) is a glycoprotein expressed on the surfaces of monocytes, neutrophils, platelets, a subpopulation of T cells, and, as described in this work, on NK cells. It is also concentrated at the junctions between endothelial cells (EC) in culture, and is expressed on continuous endothelia in all tissues. PECAM has been shown to be involved in monocyte and neutrophil transendothelial migration in vitro and in vivo. The function of PECAM in NK cell interaction with EC has never been studied. In this work, we demonstrate that ligation of PECAM on the surface of NK cells activates their beta 2 integrins. Anti-PECAM Abs added to NK cells caused a 2.5- to 4-fold increase in the binding of these cells to monolayers of EC or 3T3 cells transfected with ICAM-1, and this was inhibited by a mAb against CD18. PECAM also plays a role in NK cell transendothelial migration. Anti-PECAM Abs inhibited 50% of NK cell transmigration through resting EC in an in vitro system. The transmigration of CD56dim and CD56bright cells was inhibited equally. IFN-gamma increased NK cell transmigration; the transmigration of CD56bright cells was increased to a much greater extent than CD56dim transmigration (4-fold vs 1.5-fold). Anti-PECAM inhibited the transmigration of CD56dim cells by 30%, while that of CD56bright cells was not blocked. These studies demonstrate that PECAM-1 could play an important role in the extravasation of NK cells into tissues for constitutive surveillance and into sites of inflammation.
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the role of pecam 1 cd31 in leukocyte emigration studies in vitro and in vivo
Journal of Leukocyte Biology, 1995Co-Authors: William A MullerAbstract:Platelet/endothelial cell adhesion molecule-1 (PECAM-1, CD31) is a molecule capable of mediating both homophilic and heterophilic adhesion. It is constitutively expressed and concentrated in the lateral borders between endothelial cells and expressed on the surfaces of neutrophils, monocytes, and some T cell subsets, as well as on platelets. In a quantitative in vitro assay, monoclonal antibody against PECAM-1 or soluble recombinant PECAM-1 selectively blocked passage of both neutrophils and monocytes across the endothelial monolayer by 70-90% without interfering with the ability of these cells to bind to the apical endothelial cell surface. These regents worked whether directed against leukocyte PECAM-1 or against endothelial cell PECAM-1 and were not additive, suggesting that a homophilic interaction was occurring. In a murine model of acute inflammation, thioglycollate-induced peritonitis, a monoclonal antibody against mouse PECAM-1 blocked emigration of leukocytes into the peritoneal cavity down to background levels. Examination of peritoneal venules in these mice revealed many leukocytes in apparent contact with the endothelial surface but unable to cross the intima. Thus, PECAM-1 has a distinct role in the transendothelial migration phase of leukocyte emigration, independent of the adhesion events on the apical surface.
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platelet endothelial cell adhesion molecule 1 cd31 mediated cellular aggregation involves cell surface glycosaminoglycans
Journal of Biological Chemistry, 1993Co-Authors: Horace M Delisser, William A Muller, Steven M Albelda, Peter J. Newman, Clayton A Buck, Horng Chin YanAbstract:Platelet/endothelial cell adhesion molecule-1 (PECAM-1, CD31) is a 130-kDa integral membrane glycoprotein expressed on endothelial cells, platelets, and leukocytes. Experiments analyzing the aggregation of mouse L-cells stably transfected with full-length PECAM-1 cDNA have demonstrated that PECAM-1 is capable of mediating calcium-dependent heterophilic aggregation. In this report the ligand interactions involved in the aggregation process were studied. This aggregation was inhibited by heparin and chondroitin sulfate, but not by other glycosaminoglycans. Enzymatic removal of cell surface glycosaminoglycans confirmed a PECAM-1-glycosaminoglycan interaction and suggested that this interaction involved glycosaminoglycans on adjacent cells. PECAM-1 contains a glycosaminoglycan consensus binding sequence in the second immunoglobulin-like domain of the molecule's extracellular domain. A comparable region in the related adhesion protein N-CAM has been shown to mediate the adhesive properties of N-CAM. Cells expressing mutant PECAM-1 protein missing the second domain failed to aggregate. Synthetic peptides mimicking the consensus glycosaminoglycan binding sequence, L-K-R-E-K-N, inhibited aggregation. These results demonstrate that PECAM-1-mediated aggregation is dependent on the binding of PECAM-1 to specific glycosaminoglycans on adjacent cells via a glycosaminoglycan consensus binding sequence in the second immunoglobulin-like homology domain.
Joseph A Madri - One of the best experts on this subject based on the ideXlab platform.
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mapks erk p38 and akt can be phosphorylated by shear stress independently of platelet endothelial cell adhesion molecule 1 cd31 in vascular endothelial cells
Journal of Biological Chemistry, 2005Co-Authors: Bauer E Sumpio, Sangseob Yun, Alfredo C Cordova, Masae Haga, Jin Zhang, Yongbok Koh, Joseph A MadriAbstract:Abstract PECAM-1 (CD31) is a member of the Ig superfamily of cell adhesion molecules and is expressed on endothelial cells (EC) as several circulating blood elements including platelets, polymorphonuclear leukocytes, monocytes, and lymphocytes. PECAM-1 tyrosine phosphorylation has been observed following mechanical stimulation of EC but its role in mechanosensing is still incompletely understood. The aim of this study was to investigate the involvement of PECAM-1 in signaling cascades in response to fluid shear stress (SS) in vascular ECs. PECAM-1-deficient (KO) and PECAM-reconstituted murine microvascular ECs, 50 and 100% confluent bovine aortic EC (BAEC), and human umbilical vein EC (HUVEC) transfected with antisense PECAM-1 oligonucleotides were exposed to oscillatory SS (14 dynes/cm2) for 0, 5, 10, 30 or 60 min. The tyrosine phosphorylation level of PECAM-1 immunoprecipitated from SS-stimulated PECAM-reconstituted, but not PECAM-1-KO, murine ECs increased. Although PECAM-1 was phosphorylated in 100% confluent BAEC and HUVEC, its phosphorylation level in 50% confluent BAECs or HUVEC was not detected by SS. Likewise PECAM-1 phosphorylation was robust in the wild type and scrambled-transfected HUVEC but not in the PECAM-1 antisense-HUVEC. ERK½, p38 MAPK, and AKT were activated by SS in all cell types tested, including the PECAM-1-KO murine ECs, 50% confluent BAECs, and HUVEC transfected with antisense PECAM-1. This suggests that PECAM-1 may not function as a major mechanoreceptor for activation of MAPK and AKT in ECs and that there are likely to be other mechanoreceptors in ECs functioning to detect shear stress and trigger intercellular signals.
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altered vascular permeability and early onset of experimental autoimmune encephalomyelitis in pecam 1 deficient mice
Journal of Clinical Investigation, 2002Co-Authors: Donnasue Graesser, Monika Bruckner, Britta Engelhardt, Sandra J Davis, Anna Solowiej, Emily K Osterweil, Amy E Juedes, Nancy H Ruddle, Joseph A MadriAbstract:Platelet/endothelial cell adhesion molecule-1 (PECAM-1, CD31), a 130-kDa glycoprotein member of the Ig superfamily of transmembrane proteins, is expressed on endothelial cells, platelets, and sub- sets of leukocytes. It functions as a cell adhesion molecule as well as a scaffolding molecule capable of modulating cellular signaling pathways. In this study, using PECAM-1-deficient (KO) mice, as well as cells derived from these mice, we demonstrate that the absence of PECAM-1 expression is associ- ated with an early onset of clinical symptoms during experimental autoimmune encephalomyelitis (EAE), a mouse model for the human autoimmune disease multiple sclerosis. During EAE, mononu- clear cell extravasation and infiltration of the CNS occur at earlier time points in PECAM-KO mice than in wild-type mice. In vitro, T lymphocyte transendothelial migration across PECAM-KO endothelial cells is enhanced, regardless of expression of PECAM-1 on transmigrating T cells. Addi- tionally, cultured PECAM-KO endothelial cells exhibit prolonged permeability changes in response to histamine treatment compared with PECAM-1-reconstituted endothelial cells. Lastly, we demon- strate an exaggerated and prolonged CNS vascular permeability during the development of EAE and a delay in restoration of dermal vascular integrity following histamine challenge in PECAM-KO mice.
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platelet endothelial cell adhesion molecule 1 cd31 a scaffolding molecule for selected catenin family members whose binding is mediated by different tyrosine and serine threonine phosphorylation
Journal of Biological Chemistry, 2000Co-Authors: Neta Ilan, Larry Cheung, Emese Pinter, Joseph A MadriAbstract:Platelet-endothelial cell adhesion molecule (PECAM)-1 is a 130-kDa glycoprotein commonly used as an endothelium-specific marker. Evidence to date suggests that PECAM-1 is more than just an endothelial cell marker but is intimately involved in signal transduction pathways. This is mediated in part by phosphorylation of specific tyrosine residues within the ITAM domain of PECAM-1 and by recruitment of adapter and signaling molecules. Recently we demonstrated that PECAM-1/beta-catenin association functions to regulate beta-catenin localization and, moreover, to modulate beta-catenin tyrosine phosphorylation levels. Here we show that: 1) not only beta-catenin, but also gamma-catenin is associated with PECAM-1 in vitro and in vivo; 2) PKC enzyme directly phosphorylates purified PECAM-1; 3) PKC-derived PECAM-1 serine/threonine phosphorylation inversely correlates with gamma-catenin association; 4) PECAM-1 recruits gamma-catenin to cell-cell junctions in transfected SW480 cells; and 5) gamma-catenin may recruit PECAM-1 into an insoluble cytoskeletal fraction. These data further support the concept that PECAM-1 functions as a binder and modulator of catenins and provides a molecular mechanism for previously reported PECAM-1/cytoskeleton interactions.
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pecam 1 cd31 expression modulates bleeding time in vivo
American Journal of Pathology, 2000Co-Authors: Sepi Mahooti, Peter J. Newman, Donnasue Graesser, Sonali Patil, Gordon S Duncan, Tak W Mak, Joseph A MadriAbstract:PECAM-1 is a 130-kd member of the Ig superfamily present on endothelial cells, platelets, polymorphonuclear leukocytes, monocytes, and lymphocytes. Its expression begins early in development and persists through adulthood. PECAM-1 functions as an adhesion and signaling molecule between adjacent endothelial cells and between endothelial cells and circulating blood elements. Antibodies directed against PECAM-1 have been shown to affect angiogenesis, endothelial cell migration, and polymorphonuclear leukocyte transmigration. Furthermore, its dimerization is associated with the modulation of integrin affinity. Antibody inhibition studies suggest that PECAM-1 plays a role in modulating thrombosis; however, recent in vitro aggregation studies performed on platelets harvested from PECAM-1-deficient mice revealed no abnormalities. In this report we demonstrate prolonged in vivo bleeding times in PECAM-1-deficient mice. This abnormality was not corrected when wild-type hematopoietic precursors were engrafted into marrow-ablated PECAM-1-deficient mice. Furthermore, normal bleeding times were observed when marrow-ablated wild-type mice were engrafted with hematopoietic precursors harvested from PECAM-1-deficient mice. These studies are consistent with a role for PECAM-1 in modulating thrombosis in the vasculature, which is potentially mediated by endothelial cell PECAM-1 expression.
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platelet endothelial cell adhesion molecule 1 is phosphorylatable by c src binds src src homology 2 domain and exhibits immunoreceptor tyrosine based activation motif like properties
Journal of Biological Chemistry, 1997Co-Authors: Mark Barreuther, Sandra J Davis, Joseph A MadriAbstract:Abstract Platelet endothelial cell adhesion molecule-1 (PECAM-1) is 130-kDa member of the immunoglobulin gene superfamily that localizes to cell-cell borders of confluent endothelial cells and has been shown to play a role in the control of endothelial sheet migration and leukocyte transmigration through the endothelium. The cytoplasmic tail plays an important role in the modulation of PECAM-1 function. Mutation of tyrosine 663 or 686 in the cytoplasmic tail reduces phosphorylation and mutation of 686 is associated with a reduction in PECAM-1-mediated inhibition of cell migration (1). We have previously noted that these two tyrosine residues are surrounded by consensus sequences for Src homology 2 (SH2) domain binding (1, 2), and the experiments presented explore the potential for PECAM-1-Src and PECAM-1-SH2 domain interactions. PECAM-1 is more highly phosphorylated in endothelial cells overexpressing c-Src, and inin vitro kinase assays, c-Src can phosphorylate a glutathione S-transferase (GST)-PECAM cytoplasmic tail fusion protein. The phosphorylated fusion protein associates with the bead-bound c-Src. This association appears to be mediated by Src-SH2 domain, because PECAM-1 can be precipitated by a GST-Src-SH2 affinity matrix. The binding to the GST-Src-SH2 affinity matrix correlates directly with the level of PECAM-1 phosphorylation, because more PECAM-1 is precipitated from c-Src overexpressors and from wild-type rather than Tyr663 → Phe and Tyr686 → Phe mutant PECAM-1 expressors. Yet unidentified phosphoproteins can also be coimmunoprecipitated with wild-type but not mutant PECAM-1. Finally, we note the similarity of the PECAM-1 cytoplasmic domain sequence to the immunoreceptor tyrosine-based activation motif. Our data begin to delineate how tyrosines 663 and 686 may play a role in mediating PECAM-1 signal transduction.
Keigi Fujiwara - One of the best experts on this subject based on the ideXlab platform.
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mechanotransduction in an extracted cell model fyn drives stretch and flow elicited pecam 1 phosphorylation
Journal of Cell Biology, 2008Co-Authors: Yi Jen Chiu, Elena Mcbeath, Keigi FujiwaraAbstract:Mechanosensing followed by mechanoresponses by cells is well established, but the mechanisms by which mechanical force is converted into biochemical events are poorly understood. Vascular endothelial cells (ECs) exhibit flow- and stretch-dependent responses and are widely used as a model for studying mechanotransduction in mammalian cells. Platelet EC adhesion molecule 1 (PECAM-1) is tyrosine phosphorylated when ECs are exposed to flow or when PECAM-1 is directly pulled, suggesting that it is a mechanochemical converter. We show that PECAM-1 phosphorylation occurs when detergent-extracted EC monolayers are stretched, indicating that this phosphorylation is mechanically triggered and does not require the intact plasma membrane and soluble cytoplasmic components. Using kinase inhibitors and small interfering RNAs, we identify Fyn as the PECAM-1 kinase associated with the model. We further show that stretch- and flow-induced PECAM-1 phosphorylation in intact ECs is abolished when Fyn expression is down-regulated. We suggest that PECAM-1 and Fyn are essential components of a PECAM-1–based mechanosensory complex in ECs.
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evidence for a role of platelet endothelial cell adhesion molecule 1 in endothelial cell mechanosignal transduction is it a mechanoresponsive molecule
Journal of Cell Biology, 2002Co-Authors: Masaki Osawa, Michitaka Masuda, Ken Ichi Kusano, Keigi FujiwaraAbstract:Fluid shear stress (FSS) induces many forms of responses, including phosphorylation of extracellular signal–regulated kinase (ERK) in endothelial cells (ECs). We have earlier reported rapid tyrosine phosphorylation of platelet endothelial cell adhesion molecule-1 (PECAM-1) in ECs exposed to FSS. Osmotic changes also induced similar PECAM-1 and ERK phosphorylation with nearly identical kinetics. Because both FSS and osmotic changes should mechanically perturb the cell membrane, they might activate the same mechanosignaling cascade. When PECAM-1 is tyrosine phosphorylated by FSS or osmotic changes, SHP-2 binds to it. Here we show that ERK phosphorylation by FSS or osmotic changes depends on PECAM-1 tyrosine phosphorylation, SHP-2 binding to phospho-PECAM-1, and SHP-2 phosphatase activity. In ECs under flow, detectable amounts of SHP-2 and Gab1 translocated from the cytoplasm to the EC junction. When magnetic beads coated with antibodies against the extracellular domain of PECAM-1 were attached to ECs and tugged by magnetic force for 10 min, PECAM-1 associated with the beads was tyrosine phosphorylated. ERK was also phosphorylated in these cells. Binding of the beads by itself or pulling on the cell surface using poly-l–coated beads did not induce phosphorylation of PECAM-1 and ERK. These results suggest that PECAM-1 is a mechanotransduction molecule.
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tyrosine phosphorylation of platelet endothelial cell adhesion molecule 1 pecam 1 cd31 in mechanically stimulated vascular endothelial cells
European Journal of Cell Biology, 1997Co-Authors: Masaki Osawa, Michitaka Masuda, Noburu Harada, Rosangela Bruno Lopes, Keigi FujiwaraAbstract:Fluid flow triggers signal transducing events, modulates gene expression, and remodels cytoskeletal structures in vascular endothelial cells (ECs). However, the primary steps of mechanoreception are still unknown. We have recently reported that a glycoprotein is rapidly tyrosine-phosphorylated in bovine ECs exposed to fluid flow or osmotic shock. Here were cloned a 3.4 kb cDNA encoding this protein and found that this was bovine PECAM-1. The tyrosine-phosphorylation level of PECAM-1 immunoprecipitated from mechanically stimulated bovine or human ECs increased. The PECAM-1 phosphorylation was not induced by reagents that triggered Ca2+ mobilization in ECs. An autophosphorylatable band comigrating with c-Src was co-immunoprecipitated with anti-PECAM-1, and c-Src phosphorylated and bound to a GST fusion protein containing the PECAM-1 cytoplasmic domain. A spliced mRNA form lacking amino acid residues 703-721 in the cytoplasmic domain was also expressed in bovine ECs, c-Src neither phosphorylated nor bound to the fusion protein containing the spliced PECAM-1 cytoplasmic domain which lacked one (Tyr 713) of the six tyrosine residues in the PECAM-1 cytoplasmic domain. These results suggest that the YSEI motif containing Tyr 713 is the Src phosphorylation/binding site. Our study is the first demonstration of inducible tyrosine phosphorylation of PECAM-1 and suggests involvement of PECAM-1 and Src family kinases in the sensing/signal transduction of mechanical stimuli in ECs.