CD226 mediates platelet and megakaryocytic cell adhesion to vascular endothelial cells

Platelet adhesion to vascular endothelial cells is a pathophysiologically relevant cell-to-cell interaction. However, the mechanisms underlying this cellular interaction are incompletely understood. In search of the ligand for CD226 adhesion molecule expressed on platelets, we found that human umbilical vein endothelial cells (HUVEC) express significant amount of putative CD226 ligand. We demonstrated that thrombin-activated, but not resting, platelets bind to intact HUVEC. Anti-CD226 monoclonal antibody specifically inhibited the binding, indicating that CD226 mediates the intercellular binding between thrombin-activated platelets and HUVEC. We also demonstrated that platelet activation with thrombin induces tyrosine phosphorylation of CD226 as well as CD226-mediated platelet adhesion. Moreover, experiments using mutant transfectants suggested that the tyrosine at residue 322 of CD226 plays an important role for its adhesive function. CD226 was also expressed on primary megakaryocytes and megakaryocytic cell lines. Anti-CD226 monoclonal antibody inhibited binding of megakaryocytic cell lines to HUVEC. Taken together, these results reveal a novel mechanism for adhesion of platelets and megakaryocytic cells to vascular endothelial cells.     

Cytokine and endothelial cell adhesion molecule expression in interleukin-10-deficient mice

The objectives of this study were to quantify cytokine mRNA levels and endothelial cell adhesion molecule message and protein expression in healthy wild-type and interleukin-10-deficient (IL-10(-/-)) mice that develop spontaneous and chronic colitis. We found that colonic message levels of IL-1, IL-6, tumor necrosis factor-alpha, interferon-gamma, lymphotoxin-beta, and transforming growth factor-beta were elevated in colitic mice 10- to 35-fold compared with their healthy wild-type controls. In addition, colonic message levels of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and mucosal addressin cell adhesion molecule-1 (MAdCAM-1) were found to be increased 10-, 5-, and 23-fold, respectively, in colitic IL-10(-/-) mice compared with their wild-type controls. Immunoradiolabeling as well as immunohistochemistry revealed large and significant increases in vascular surface expression of colonic ICAM-1, VCAM-1, and MAdCAM-1 in the mucosa as well as the submucosa of the colons of colitic mice. These data are consistent with the hypothesis that deletion of IL-10 results in the sustained production of proinflammatory cytokines, leading to the upregulation of adhesion molecules and infiltration of mononuclear and polymorphonuclear leukocytes into the cecal and colonic interstitium.     

Pro-oxidant role of heme oxygenase in mediating glucose-induced endothelial cell damage

Oxidative damage to the vascular endothelial cells may play a crucial role in mediating glucose-induced cellular dysfunction in chronic diabetic complications. The present study was aimed at elucidating the role of glucose-induced alteration of highly inducible heme oxygenase (HO) in mediating oxidative stress in the vascular endothelial cells. We have also investigated the interaction between HO and the nitric oxide (NO) system, and its possible role in alteration of other vasoactive factors. Human umbilical vein endothelial cells (HUVECs) were exposed to low (5mmol/l) and high (25mmol/l) glucose levels. In order to determine the role of HO in endothelial dysfunction and to elucidate a possible interaction between the HO and NO systems, cells were exposed to HO inducer (hemin, 10 micromol/l), HO antagonist (SnPPIX, 10 micromol/l), and NO synthase blocker (L-NAME, 200 micromol/l) with or without NO donor (arginine, 1 mmol/l). mRNA expression of HO and NO isoforms was measured by real time RT-PCR. HO activity was measured by bilirubin production and cellular oxidative stress was assessed by 8-hydroxy-2'-deoxyguanosine (8-OHdG) and nitrotyrosine staining. We also determined the expression of vasoactive factors, endothelin-1 (ET-1) and vascular endothelial growth factor (VEGF). In the endothelial cells, glucose caused upregulation of HO-1 expression and increased HO activity. A co-stimulatory relationship between HO and NO was observed. Increased HO activity also associated with oxidative DNA and protein damage in the endothelial cells. Furthermore, increased HO activity augmented mRNA expression of vasoactive factors, ET-1 and VEGF. These data suggest that HO by itself and via elaboration of other vasoactive factors may cause endothelial injury and functional alteration. These findings are of importance in the context of chronic diabetic complications.     

Essential role of Src suppressed C kinase substrates in endothelial cell adhesion and spreading

Integrin-mediated substrate adhesion of endothelial cells leads to dynamic rearrangement of the actin cytoskeleton. Protein kinase C (PKC) stimulates reorganization of microfilaments and adhesion, but the mechanism by which this occurs is unknown. Src suppressed C kinase substrate (SSeCKS) is a PKC substrate that may play an important role in regulating actin cytoskeleton. We found that SSeCKS was localized to focal adhesion sites soon after cell adhesion and that SSeCKS translocated from the membrane to the cytosol during the process of cell spreading. Using small interfering RNAs specific to SSeCKS, we show that RPMVEC cells in which SSeCKS expression was inhibited reduce adhesion and spread on LN through blocking the formation of actin stress fibers and focal adhesions. These results demonstrated SSeCKS modulate endothelial cells adhesion and spreading by reorganization of the actin cytoskeleton.     

Sphingosine 1-phosphate induces platelet/endothelial cell adhesion molecule-1 phosphorylation in human endothelial cells through cSrc and Fyn

Sphingosine 1-phosphate (S1P) is a multifunctional phospholipid which acts through a specific family of G protein-coupled receptors. Platelet/endothelial cell adhesion molecule-1 (PECAM-1) form trans-homophilic binding at lateral cell border. Upon stimulation, its cytoplasmic tyrosine residues could be phosphorylated and interact with various downstream signaling molecules. In this study, we demonstrated that S1P induced PECAM-1 tyrosine phosphorylation in human umbilical cord vein cells (HUVECs). By pharmacological inhibitors, it was suggested that G(i) and Src family kinases were involved in PECAM-1 phosphorylation. Moreover, cSrc and Fyn siRNA significantly suppressed S1P-induced PECAM-1 phosphorylation. These results suggested that S1P-induced PECAM-1 phosphorylation through G(i) and subsequent cSrc and Fyn. Our findings provide further understanding of S1P and PECAM-1 signaling as well as their functions in endothelial cells.     

Differential modulation of cadherin-mediated cell-cell adhesion by platelet endothelial cell adhesion molecule-1 isoforms through activation of extracellular regulated kinases

The role of platelet endothelial cell adhesion molecule-1 (PECAM-1) in endothelial cell-cell interactions and its contribution to cadherin-mediated cell adhesion are poorly understood. Such studies have been difficult because all known endothelial cells express PECAM-1. We have used Madin-Darby canine kidney (MDCK) cells as a model system in which to evaluate the role of PECAM-1 isoforms that differ in their cytoplasmic domains in cell-cell interactions. MDCK cells lack endogenous PECAM-1 but form cell-cell junctions similar to those of endothelial cells, in which PECAM-1 is concentrated. MDCK cells were transfected with two isoforms of murine PECAM-1, Delta15 and Delta14&15, the predominant isoforms expressed in vivo. Expression of the Delta15 isoform resulted in apparent dedifferentiation of MDCK cells concomitant with the loss of adherens junctions, down-regulation of E-cadherin, alpha- and beta-catenin expression, and sustained activation of extracellular regulated kinases. The Delta15 isoform was not concentrated at cell-cell contacts. In contrast, the Delta14&15 isoform localized to sites of cell-cell contact and had no effect on MDCK cell morphology, cadherin/catenin expression, or extracellular regulated kinase activity. Thus, the presence of exon 14 in the cytoplasmic domain of PECAM-1 has dramatic effects on the ability of cells to maintain adherens junctions and an epithelial phenotype. Therefore, changes in the expression of exon 14 containing PECAM-1 isoforms, which we have observed during development, may have profound functional consequences.     

Human cytomegalovirus infection of endothelial cells triggers platelet adhesion and aggregation

Human cytomegalovirus (HCMV) is an opportunistic pathogen that has been implicated in the pathogenesis of vascular diseases. HCMV infection of endothelial cells may lead to vascular damage in vitro, and acute-phase HCMV infection has been associated with thrombosis. We hypothesized that viral infection of endothelial cells activates coagulation cascades and contributes to thrombus formation and acute vascular catastrophes in patients with atherosclerotic disease. To assess the effects of HCMV on thrombogenesis, we examined the adhesion and aggregation of blood platelets to uninfected and HCMV-infected endothelial cells. At 7 days after infection, platelet adherence and aggregation were greater in infected than in uninfected cultures (2,000 platelets/100 cells and 225 +/- 15 [mean +/- standard error of the mean] aggregates/five microscopic fields versus 100 platelets/100 cells and no aggregates). von Willebrand factor (vWF), ICAM-1, and VCAM-1 but not collagen IV, E-selectin, P-selectin, CD13, and CD31 were expressed at higher levels on infected cells than on uninfected cells. Platelet aggregation was inhibited by blocking of platelet GPIb (with blocking antibodies) or GPIIb/IIIa (with ReoPro) or by blocking of vWF (with polyclonal antibodies to vWF). Furthermore, blocking of vWF, platelet GPIb, and ICAM-1 but not of the endothelial cell marker CD13, alpha(5)beta(3)-integrin, or HCMV glycoprotein B reduced platelet adherence to infected cells by 75% +/- 5%, 74% +/- 5%, or 18% +/- 5%, respectively. The increased thrombogenicity was dependent on active virus replication and could be inhibited by foscarnet and ganciclovir; these results suggest that a late viral gene may be mediating this phenomenon, which may contribute to vascular catastrophes in patients with atherosclerotic disease.     

Identification of Fer tyrosine kinase localized on microtubules as a platelet endothelial cell adhesion molecule-1 phosphorylating kinase in vascular endothelial cells

Platelet endothelial adhesion molecule-1 (PECAM-1) is a part of intercellular junctions and triggers intracellular signaling cascades upon homophilic binding. The intracellular domain of PECAM-1 is tyrosine phosphorylated upon homophilic engagement. However, it remains unclear which tyrosine kinase phosphorylates PECAM-1. We sought to isolate tyrosine kinases responsible for PECAM-1 phosphorylation and identified Fer as a candidate, based on expression cloning. Fer kinase specifically phosphorylated PECAM-1 at the immunoreceptor tyrosine-based inhibitory motif. Notably, Fer induced tyrosine phosphorylation of SHP-2, which is known to bind to the immunoreceptor tyrosine-based inhibitory motif of PECAM-1, and Fer also induced tyrosine phosphorylation of Gab1 (Grb2-associated binder-1). Engagement-dependent PECAM-1 phosphorylation was inhibited by the overexpression of a kinase-inactive mutant of Fer, suggesting that Fer is responsible for the tyrosine phosphorylation upon PECAM-1 engagement. Furthermore, by using green fluorescent protein-tagged Fer and a time-lapse fluorescent microscope, we found that Fer localized at microtubules in polarized and motile vascular endothelial cells. Fer was dynamically associated with growing microtubules in the direction of cell-cell contacts, where p120catenin, which is known to associate with Fer, colocalized with PECAM-1. These results suggest that Fer localized on microtubules may play an important role in phosphorylation of PECAM-1, possibly through its association with p120catenin at nascent cell-cell contacts.     

Soluble platelet/endothelial cell adhesion molecule (sPECAM)-1 is increased in polycystic ovary syndrome and related to endothelial dysfunction

Striking evidence indicates endothelial impairment in polycystic ovary syndrome (PCOS) but the mechanisms linking PCOS status to cardiovascular risk remain elusive. Platelet/endothelial cell adhesion molecule (PECAM)-1 is a soluble (s) signaling molecule involved in inflammation and angiogenesis with predictive value for endothelial dysfunction in patients at risk. In a prospective, controlled study, sPECAM-1 levels and the relationships to metabolic, inflammatory and vascular PCOS traits were evaluated in 26 patients and 29-age- and body mass index-matched controls. To assess endothelial injury, carotid artery intimae-media thickness (CIMT) and brachial artery flow-mediated vasodilatation (FMD) were employed. Of the 26 women with PCOS, 25 completed a six-month metformin combined with ethinylestradiol 0.3?mg/drospirenone 3?mg therapy. Soluble PECAM-1 levels were increased in PCOS (p?=?0.018 vs. Controls) and significantly decreased at follow-up (p?=?0.0002). Smoking and weight had no effect on sPECAM-1 dynamics. In both univariate and multivariate analysis, basal sPECAM-1 was inversely related to FMD (r?=?-0.311, p?=?0.021) but not CIMT. To conclude, sPECAM-1 is increased in PCOS, an effect reversed by combined metformin and anti-androgenic contraceptive therapy. Elevated sPECAM-1 contributes to endothelial dysfunction however further studies are inquired to assess its relevance as biomarker and potential therapeutic target in PCOS.     

Eosinophil tissue recruitment to sites of allergic inflammation in the lung is platelet endothelial cell adhesion molecule independent

Platelet endothelial cell adhesion molecule (PECAM or CD31) is a cell adhesion molecule expressed on circulating leukocytes and endothelial cells that plays an important role in mediating neutrophil and monocyte transendothelial migration in vivo. In this study, we investigated whether eosinophils, like neutrophils and monocytes, utilize PECAM for tissue recruitment to sites of allergic inflammation in vivo. Eosinophils express similar levels of PECAM as neutrophils as assessed by FACS analysis. RT-PCR studies demonstrate that eosinophils like neutrophils express the six extracellular domains of PECAM. Eosinophils exhibit homophilic binding to recombinant PECAM as assessed in a single-cell micropipette adhesion assay able to measure the biophysical strength of adhesion of eosinophils to recombinant PECAM. The strength of eosinophil adhesion to recombinant PECAM is the same as that of neutrophil binding to recombinant PECAM and can be inhibited with an anti-PECAM Ab. Although eosinophils express functional PECAM, anti-PECAM Abs did not inhibit bronchoalveolar lavage eosinophilia, lung eosinophilia, and airway hyperreactivity to methacholine in a mouse model of OVA-induced asthma in vivo. Thus, in contrast to studies that have demonstrated that neutrophil and monocyte tissue recruitment is PECAM dependent, these studies demonstrate that eosinophil tissue recruitment in vivo in this model is PECAM independent.     

Change in platelet endothelial cell adhesion molecule-1 immunoreactivity in the dentate gyrus in gerbils fed a folate-deficient diet

Folate deficiency increases stroke risk. We examined whether folate deficiency affects platelet endothelial cell adhesion molecule-1 (PECAM-1), which is an immunoglobulin-associated cell adhesion molecule and mediates the final common pathway of neutrophil transendothelial migration, in blood vessels in the gerbil dentate gyrus after transient forebrain ischemia. Gerbils were exposed to a folic acid-deficient diet (FAD) for 3 months and then subjected to common carotid artery occlusion for 5 min. In the control diet (CD)- and FAD-treated sham-operated groups, weak PECAM-1 immunoreactivity was detected in the blood vessels located in the dentate gyrus. PECAM-1 immunoreactivity in both groups was increased by 4 days after ischemic insult. PECAM-1 immunoreactivity in the FAD-treated group was twice as high that in the CD-treated-sham-operated group 4 days after ischemic insult. Western blot analyses showed that the change patterns in PECAM-1 protein levels in the dentate gyrus in both groups after ischemic insult were similar to changes in PECAM-1 immunohistochemistry in the ischemic dentate gyrus. Our results suggest that folate deficiency enhances PECAM-1 in the dentate gyrus induced by transient ischemia.     

Ultrastructural localization of platelet endothelial cell adhesion molecule (PECAM-1, CD31) in vascular endothelium.

The distribution of platelet endothelial cell adhesion molecule (PECAM-1, CD31) in vascular endothelium has been disputed. Originally reported to be highly concentrated at interendothelial cell contacts, recent studies have claimed that CD31 is distributed evenly over the entire endothelial cell surface. We re-investigated this question with two different murine anti-CD31 antibodies (MEC 13.3 and M-20), using a pre-embedding immunonanogold electron microscopic procedure that allowed precise label quantitation. MEC 13.3 reacted strongly with the luminal and abluminal plasma membranes of the endothelial cells lining microvessels in normal tissues and in angiogenic vessels induced by a tumor and vascular endothelial growth factor (VEGF-A164). Lateral plasma membranes were significantly less labeled. Conversely, M-20 strongly labeled the cytoplasmic face of the lateral plasma membranes of endothelial cells, although sparing specialized junctions, and only weakly labeled the luminal and abluminal plasma membranes. Both antibodies stained a significant minority of vesicles and vacuoles comprising the vesiculovacuolar organelle (VVO). Neither antibody was reactive in CD31-null mice. We conclude that CD31 is distributed over the entire endothelial cell surface, exclusive of specialized junctions, and in VVOs, but is not equally accessible to different antibodies in all locations.     

Integrin activation by regulated dimerization and oligomerization of platelet endothelial cell adhesion molecule (PECAM)-1 from within the cell

Platelet endothelial cell adhesion molecule (PECAM)-1 is a 130-kD transmembrane glycoprotein having six Ig homology domains within its extracellular domain and an immunoreceptor tyrosine-based inhibitory motif within its cytoplasmic domain. Previous studies have shown that addition of bivalent anti-PECAM-1 mAbs to the surface of T cells, natural killer cells, neutrophils, or platelets result in increased cell adhesion to immobilized integrin ligands. However, the mechanism by which this occurs is not clear, and it is possible that anti-PECAM-1 mAbs elicit this effect by simply sequestering PECAM-1, via antibody-induced patching and capping, away from stimulatory receptors that it normally regulates. To determine whether dimerization or oligomerization of PECAM-1 directly initiates signal transduction pathways that affect integrin function in an antibody-independent manner, stable human embryonic kidney-293 cell lines were produced that expressed chimeric PECAM-1 cDNAs containing one or two FK506-binding protein (FKBP) domains at their COOH terminus. Controlled dimerization initiated by addition of the bivalent, membrane-permeable FKBP dimerizer, AP1510, nearly doubled homophilic binding capacity, whereas AP1510-induced oligomers favored cis PECAM-1/PECAM-1 associations within the plane of the plasma membrane at the expense of trans homophilic adhesion. Importantly, AP1510-induced oligomerization resulted in a marked increase in both adherence and spreading of PECAM/FKBP-2-transfected cells on immobilized fibronectin, a reaction that was mediated by the integrin alpha(5)beta(1). These data demonstrate that signals required for integrin activation can be elicited by clustering of PECAM-1 from inside the cell, and suggest that a dynamic equilibrium between PECAM-1 monomers, dimers, and oligomers may control cellular activation signals that influence the adhesive properties of vascular cells that express this novel member of the immunoreceptor tyrosine-based inhibitory motif family of regulatory receptors.     

Transgenic mice expressing different levels of soluble platelet/endothelial cell adhesion molecule-IgG display distinct inflammatory phenotypes.

Platelet/endothelial cell adhesion molecule-1 (PECAM-1, CD31), expressed on the surfaces of leukocytes and concentrated in the junctions between endothelial cells plays an important role in transendothelial migration of neutrophils and monocytes. Soluble recombinant PECAM-IgG injected i.v. into mice blocks acute leukocyte emigration by 80%. To study the role of PECAM in models of chronic inflammation, we generated transgenic mice constitutively expressing soluble full-length murine PECAM as an IgG chimera. Three founder lines expressed this transgene and constitutively secreted murine PECAM-IgG into the plasma where it was maintained at characteristic concentrations for each line. All mice had similar hematologic profiles to wild-type littermates and were healthy when maintained in the standard laboratory animal facility. Both the leukocytes and the endothelium of mice of all transgenic lines expressed the same levels of endogenous PECAM-1 as wild-type littermates. Similarly, there were no detectable differences in the expression of several other common leukocyte and endothelial cell adhesion molecules. Mice that produced moderate (10-20 microg/ml) concentrations of PECAM-IgG demonstrated a severely blunted acute inflammatory response, despite mobilizing appropriate numbers of circulating leukocytes. Surprisingly, mice that constitutively produced high (400-1,000 microg/ml) concentrations of PECAM-IgG were unresponsive to its anti-inflammatory effects. This is the first demonstration that a soluble form of a cell adhesion molecule can be stably expressed and retain efficacy in vivo over prolonged periods. This approach is applicable to many other extracellular molecules. However, the plasma concentrations of such constitutively produced inhibitors may greatly influence the resulting phenotype.     

Association between IL10, IL10RA, and IL10RB SNPs and ischemic stroke with hypertension in Korean population

The pathogenesis of stroke is associated with the immune and inflammatory responses. Cytokines, such as interleukin 10 (IL10), play an important role in the process of inflammation. To investigate whether IL10, IL10RA, and IL10RB polymorphisms are associated with the risk of ischemic stroke (IS), selected two IL10 SNPs (rs1518111 and rs1554286), three IL10RA SNPs (rs2256111, rs4252243, and rs2228054), and two IL10RB SNPs (rs999788 and rs2834167) were analyzed in 120 patients with IS and 285 control subjects. All IS patients were classified into the clinical subgroups, according to the levels of blood pressure (hypertension, present and absent), fasting plasma glucose (diabetes mellitus, present and absent), and lipids (dyslipidemia, present and absent). SNPStats and SPSS 18.0 program were used to obtain the odds ratios, 95 % confidence intervals, and P values. Multiple logistic regression models (codominant1, codominant2, dominant, recessive, and log-additive models) were performed to analyze the genetic data. Seven polymorphisms were not associated with the IS, but showed significant associations with hypertension, in the risk of IS. These results suggest that the IL10, IL10RA, and IL10RB genes may be contributed to the hypertension in the risk of IS in the Korean population.