Differential movements of VE-cadherin and PECAM-1 during transmigration of polymorphonuclear leukocytes through human umbilical vein endothelium

Most existing evidence regarding junction protein movements during transendothelial migration of leukocytes comes from taking postfixation snap shots of the transendothelial migration process that happens on a cultured endothelial monolayer. In this study, we used junction protein-specific antibodies that did not interfere with the transendothelial migration to examine the real-time movements of vascular endothelial-cadherin (VE-cadherin) and platelet/endothelial cell adhesion molecule-1 (PECAM-1) during transmigration of polymorphonuclear leukocytes (PMNs) either through a cultured endothelial monolayer or through the endothelium of dissected human umbilical vein tissue. In either experimental model system, both junction proteins showed relative movements, not transient disappearance, at the PMN transmigration sites. VE-cadherin moved away to different ends of the transmigration site, whereas PECAM-1 opened to surround the periphery of a transmigrating PMN. Junction proteins usually moved back to their original positions when the PMN transmigration process was completed in less than 2 minutes. The relative positions of some junction proteins might rearrange to form a new interendothelial contour after PMNs had transmigrated through multicellular corners. Although transmigrated PMNs maintained good mobility, they only moved laterally underneath the vascular endothelium instead of deeply into the vascular tissue. In conclusion, our results obtained from using either cultured cells or vascular tissues showed that VE-cadherin-containing adherent junctions were relocated aside, not opened or disrupted, whereas PECAM-1-containing junctions were opened during PMN transendothelial migration.     

PECAM-1-dependent neutrophil transmigration is independent of monolayer PECAM-1 signaling or localization

Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31), a tyrosine phosphoprotein highly expressed on endothelial cells and leukocytes, is an important component in the regulation of neutrophil transendothelial migration. Engagement of endothelial PECAM-1 activates tyrosine phosphorylation events and evokes prolonged calcium transients, while homophilic engagement of neutrophil PECAM-1 activates leukocyte beta-integrins. Although PECAM-1 modulates polymorphoneutrophil transmigration via homophilic PECAM-1-PECAM-1 interaction, the mechanisms underlying endothelial PECAM-1 function are unknown. Proposed mechanisms include (1) formation of a haptotactic gradient that "guides" neutrophils to the cell-cell border, (2) service as a "passive ligand" for neutrophil PECAM-1, ultimately mediating activation of neutrophil beta integrins, (3) regulation of endothelial calcium influx, and (4) mediation of SH2 protein association, and/or (5) catenin and non-SH2 protein interaction. Utilizing PECAM-1-null "model" endothelial cells (REN cells), we developed a neutrophil transmigration system to study PECAM-1 mutations that specifically disrupt PECAM-1-dependent signaling and/or PECAM-1 cell localization. We report that interleukin-1 beta (IL-1 beta) elicits PECAM-1-dependent transmigration that requires homophilic PECAM-PECAM-1 engagement, but not heterophilic neutrophil PECAM-1 interactions, and is intercellular adhesion molecule-1 dependent. Conversely, whereas IL-8 and leukotriene-B(4)-mediated transmigration is PECAM-1-independent, PECAM-1 and IL-8-dependent transmigration represent separable and additive components of cytokine-induced transmigration. Surprisingly, neither monolayer PECAM-1-regulated calcium signaling, cell border localization, nor the PECAM-1 cytoplasmic domain was required for monolayer PECAM-1 regulation of neutrophil transmigration. We conclude that monolayer (endothelial cell) PECAM-1 functions as a passive homophilic ligand for neutrophil PECAM-1, which after engagement leads to neutrophil signal transduction, integrin activation, and ultimately transmigration in a stimulus-specific manner.     

Endothelial function and hemostasis

The vascular endothelium influences not only the three classically interacting components of hemostasis: the vessel, the blood platelets and the clotting and fibrinolytic systems of plasma, but also the natural sequelae: inflammation and tissue repair. Two principal modes of endothelial behaviour may be differentiated, best defined as an anti- and a prothrombotic state. Under physiological conditions endothelium mediates vascular dilatation (formation of NO, PGI2, adenosine, hyperpolarizing factor), prevents platelet adhesion and activation (production of adenosine, NO and PGI2, removal of ADP), blocks thrombin formation (tissue factor pathway inhibitor, activation of protein C via thrombomodulin, activation of antithrombin III) and mitigates fibrin deposition (t- and scuplasminogen activator production). Adhesion and transmigration of inflammatory leukocytes are attenuated, e.g. by NO and IL-10, and oxygen radicals are efficiently scavenged (urate, NO, glutathione, SOD). When the endothelium is physically disrupted or functionally perturbed by postischemic reperfusion, acute and chronic inflammation, atherosclerosis, diabetes and chronic arterial hypertension, then completely opposing actions pertain. This prothrombotic, proinflammatory state is characterised by vaso-constriction, platelet and leukocyte activation and adhesion (externalization, expression and upregulation of von Willebrand factor, platelet activating factor, P-selectin, ICAM-1, IL-8, MCP-1, TNF alpha, etc.), promotion of thrombin formation, coagulation and fibrin deposition at the vascular wall (expression of tissue factor, PAI-1, phosphatidyl serine, etc.) and, in platelet-leukocyte coaggregates, additional inflammatory interactions via attachment of platelet CD40-ligand to endothelial, monocyte and B-cell CD40. Since thrombin formation and inflammatory stimulation set the stage for later tissue repair, complete abolition of such endothelial responses cannot be the goal of clinical interventions aimed at limiting procoagulatory, prothrombotic actions of a dysfunctional vascular endothelium.     

Roles of angiopoietin-1 and angiopoietin-2 on airway microvascular permeability in asthmatic patients

BACKGROUND: Vascular endothelial growth factor (VEGF) increases microvascular permeability. Recently, considerable attention has been devoted to the physiologic roles of angiopoietin-1 and angiopoietin-2 as regulatory factors of VEGF. This study was designed to examine the roles of angiopoietin-1 and angiopoietin-2 in controlling airway microvascular permeability in asthma. METHODS: Levels of these angiogenic factors and airway vascular permeability index were examined in 30 asthmatics and 12 control subjects. After 2-week run-in period, all asthmatics were randomly assigned to receive fluticasone propionate (400 mug/d) or montelukast (10 mg) for 12 weeks. RESULTS: VEGF, angiopoietin-1, and angiopoietin-2 levels in induced sputum were significantly higher in asthmatics than in control subjects. We found an inverse correlation between angiopoietin-1 level and vascular permeability index in asthmatics, while there was a positive correlation between angiopoietin-2 level and that index. VEGF and angiopoietin-1 levels were significantly decreased after fluticasone therapy, while VEGF and angiopoietin-2 levels were significantly decreased after montelukast therapy. Although VEGF levels after treatment were different between two groups, vascular permeability index in the montelukast group was the same level as that in the fluticasone group. Moreover, improvement in vascular permeability index after fluticasone therapy was inversely correlated with decrease in angiopoietin-1 level, while that after montelukast therapy was positively correlated with decrease in angiopoietin-2 level. CONCLUSIONS: Angiopoietin-1 and angiopoietin-2 play complementary and coordinated roles in regulating microvascular permeability stimulated by VEGF in asthma. Combination of corticosteroids with leukotriene antagonists might effectively improve plasma leakage and provide a new strategy in treating bronchial asthma.     

JAM-A mediates neutrophil transmigration in a stimulus-specific manner in vivo: evidence for sequential roles for JAM-A and PECAM-1 in neutrophil transmigration

Junctional adhesion molecule-A (JAM-A) is a transmembrane protein expressed at tight junctions of endothelial and epithelial cells and on the surface of platelets and leukocytes. The role of JAM-A in leukocyte transmigration in vivo was directly investigated by intravital microscopy using both a JAM-A-neutralizing monoclonal antibody (mAb) (BV-11) and JAM-A-deficient (knockout [KO]) mice. Leukocyte transmigration (but not adhesion) through mouse cremasteric venules as stimulated by interleukin 1beta (IL-1beta) or ischemia/reperfusion (I/R) injury was significantly reduced in wild-type mice treated with BV-11 and in JAM-A KO animals. In contrast, JAM-A blockade/genetic deletion had no effect on responses elicited by leukotriene B(4) (LTB(4)) or platelet-activating factor (PAF). Furthermore, using a leukocyte transfer method and mice deficient in endothelial-cell JAM-A, evidence was obtained for the involvement of endothelial-cell JAM-A in leukocyte transmigration mediated by IL-1beta. Investigation of the functional relationship between JAM-A and PECAM-1 (CD31) determined that dual blockade/deletion of these proteins does not lead to an inhibitory effect greater than that seen with blockade/deletion of either molecule alone. The latter appeared to be due to the fact that JAM-A and PECAM-1 can act sequentially to mediate leukocyte migration through venular walls in vivo.     

Breakdown of paraendothelial barrier function during Marburg virus infection is associated with early tyrosine phosphorylation of platelet endothelial cell adhesion molecule-1

Marburg virus (MARV) infection often causes fulminant shock due to pathologic immune responses and alterations of the vascular system. Cytokines released from virus-infected monocytes/macrophages provoke endothelial activation and vascular hyperpermeability and contribute to the development of shock. Tyrosine phosphorylation of cell-junction proteins is important for the regulation of paraendothelial barrier function. We showed that mediators released from MARV-infected monocytes/macrophages, as well as recombinant tumor necrosis factor (TNF)- alpha /H2O2 and interferon (IFN)- gamma , caused tyrosine phosphorylation of platelet endothelial cell adhesion molecule-1 (PECAM-1) but not of the vascular endothelial (VE) cadherin/catenin complex proteins. Tyrosine phosphorylation of PECAM-1 was associated with delayed opening of interendothelial junctions. Interestingly, we observed an early increase in water permeability in response to TNF- alpha /H2O2 that was not due to an opening of the interendothelial junctions. These data indicate 2 distinct mechanisms for the TNF- alpha /H2O2-mediated decrease in endothelial barrier function involving tyrosine phosphorylation of PECAM-1 but not requiring tyrosine phosphorylation of VE-cadherin or catenin proteins.     

Ca2+ signaling, TRP channels, and endothelial permeability

Increased endothelial permeability is the hallmark of inflammatory vascular edema. Inflammatory mediators that bind to heptahelical G protein-coupled receptors trigger increased endothelial permeability by increasing the intracellular Ca2+ concentration ([Ca2+]i). The rise in [Ca2+]i activates key signaling pathways that mediate cytoskeletal reorganization (through myosin-light-chain-dependent contraction) and the disassembly of VE-cadherin at the adherens junctions. The Ca2+-dependent protein kinase C (PKC) isoform PKCalpha plays a crucial role in initiating endothelial cell contraction and disassembly of VE-cadherin junctions. The increase in [Ca2+]i induced by inflammatory agonists such as thrombin and histamine is achieved by the generation of inositol 1,4,5-trisphosphate (IP3), activation of IP3-receptors, release of stored intracellular Ca2+, and Ca2+ entry through plasma membrane channels. IP3-sensitive Ca2+-store depletion activates plasma membrane cation channels (i.e., store-operated cation channels [SOCs] or Ca2+ release-activated channels [CRACs]) to cause Ca2+ influx into endothelial cells. Recent studies have identified members of Drosophila transient receptor potential (TRP) gene family of channels that encode functional SOCs in endothelial cells. These studies also suggest that the canonical TRPC homologue TRPC1 is the predominant isoform expressed in human vascular endothelial cells, and is the essential component of the SOC in this cell type. Further, evidence suggests that the inflammatory cytokine tumor necrosis factor-alpha can induce the expression of TRPC1 in human vascular endothelial cells signaling via the nuclear factor-kappaB pathway. Increased expression of TRPC1 augments Ca2+ influx via SOCs and potentiates the thrombin-induced increase in permeability in human vascular endothelial cells. Deletion of the canonical TRPC homologue in mouse, TRPC4, caused impairment in store-operated Ca2+ current and Ca2+-store release-activated Ca2+ influx in aortic and lung endothelial cells. In TRPC4 knockout (TRPC4-/-) mice, acetylcholine-induced endothelium-dependent smooth muscle relaxation was drastically reduced. In addition, TRPC4-/- mouse-lung endothelial cells exhibited lack of actin-stress fiber formation and cell retraction in response to thrombin activation of protease-activated receptor-1 (PAR-1) in endothelial cells. The increase in lung microvascular permeability in response to PAR-1 activation was inhibited in TRPC4-/- mice. These results indicate that endothelial TRP channels such as TRPC1 and TRPC4 play an important role in signaling agonist-induced increases in endothelial permeability.     

CD44 regulates vascular endothelial barrier integrity via a PECAM-1 dependent mechanism

Vascular integrity is a critical parameter in normal growth and development. Loss of appropriate vascular barrier function is present in various immune- and injury-mediated pathological conditions. CD44 is an adhesion molecule expressed by multiple cell types, including endothelial cells (EC). The goal of the present study was to examine how loss of CD44 affected vascular permeability. Using C57BL/6 WT and CD44-KO mice, we found no significant permeability to Evan’s Blue in either strain at baseline. However, there was significantly increased histamine-induced permeability in CD44-deficient mice compared to WT counterparts. Similar results were observed in vitro, where CD44-deficient endothelial monolayers were also impermeable to 40kD-FITC dextran in the absence of vasoactive challenge, but exhibited enhanced and prolonged permeability following histamine. However, CD44-KO monolayers have reduced baseline barrier strength by electrical resistance, which correlated with increased permeability, at baseline, to smaller molecular weight 4-kD FITC-dextran, suggesting weakly formed endothelial junctions. The CD44-KO EC displayed several characteristics consistent with impaired barrier function/dysfunctional EC junctions, including differential expression, phosphorylation, and localization of endothelial junction proteins, increased matrix metalloprotease expression, and altered cellular morphology. Reduced platelet endothelial cell adhesion molecule-1 (PECAM-1) expression by CD44-KO EC in vivo and in vitro was also observed. Reconstitution of murine CD44 or PECAM-1 restored these defects to near WT status, suggesting CD44 regulates vascular permeability and integrity through a PECAM-1 dependent mechanism.     

Role of protein kinase Czeta in thrombin-induced endothelial permeability changes: inhibition by angiopoietin-1

Endothelial cell leakiness is regulated by mediators such as thrombin, which promotes endothelial permeability, and anti-inflammatory agents, such as angiopoietin-1. Here we define a new pathway involved in thrombin-induced permeability that involves the atypical protein kinase C isoform, PKCzeta. Chemical inhibitor studies implicated the involvement of an atypical PKC isoform in thrombin-induced permeability changes in human umbilical vein endothelial cells. Thrombin stimulation resulted in PKCzeta, but not the other atypical PKC isoform, PKClambda, translocating to the membrane, an event known to be critical to enzyme activation. The involvement of PKCzeta was confirmed by overexpression of constitutively active PKCzeta, resulting in enhanced basal permeability. Dominant-negative PKCzeta prevented the thrombin-mediated effects on endothelial cell permeability and inhibited thrombin-induced activation of PKCzeta. Rho activation does not appear to play a role, either upstream or downstream of PKCzeta, as C3 transferase does not block thrombin-induced PKCzeta activation and dominant-negative PKCzeta does not block thrombin-induced Rho activation. Finally, we show that angiopoietin-1 inhibits thrombin-induced PKCzeta activation, Rho activation, and Ca(++) flux, thus demonstrating that the powerful antipermeability action of angiopoietin-1 is mediated by its action on a number of signaling pathways induced by thrombin and implicated in permeability changes.     

Reversible opening of the blood-brain barrier by anti-bacterial antibodies.

The leukocyte adhesion molecule CR3 (CD11b/CD18, Mac-1) promotes leukocyte transmigration into tissues by engaging an unknown cognate ligand on the surface of vascular endothelial cells. Filamentous hemagglutinin (FHA), an adhesin of the bacterium Bordetella pertussis, binds to CR3. We hypothesized that FHA mimics the native ligand for the CR3 integrin on endothelial cells and predicted that anti-FHA antibodies should bind to endothelial cells, interfere with leukocyte recruitment, and induce endothelial permeability. Anti-FHA monoclonal antibodies bound to cerebral microvessels in sections from human brain and upon intravenous injection into rabbits. Antibody binding correlated with the ability to recognize two polypeptides in extracts of human cerebral vessels that were also bound by CD18. In vivo, antibody binding not only interfered with transmigration of leukocytes into cerebrospinal fluid but also induced a dose-dependent reversible increase in blood-brain barrier permeability sufficient to improve delivery of intravenously administered therapeutic agents to brain parenchyma.     

Regulation of platelet biology by platelet endothelial cell adhesion molecule-1

Platelet endothelial cell adhesion molecule-1 (PECAM-1), an immunoreceptor tyrosine-based inhibitory motif containing receptor, plays diverse and apparently contradictory roles in regulating the response of platelets to stimuli; inhibiting platelet response to immunoreceptor tyrosine-based activation motif and G protein-coupled receptor signalling following stimulation with collagen, adenosine diphosphate, and thrombin, as well as enhancing integrin outside-in signalling. These dual, and opposing, roles suggest an important and complex role for PECAM-1 in orchestrating platelet response to vascular damage. Indeed, during thrombus formation, the influence of PECAM-1 on the multiple signalling pathways combines leading to a relatively large inhibitory effect on thrombus formation.     

Regulation of platelet biology by platelet endothelial cell adhesion molecule-1

Platelet endothelial cell adhesion molecule-1 (PECAM-1), an immunoreceptor tyrosine-based inhibitory motif containing receptor, plays diverse and apparently contradictory roles in regulating the response of platelets to stimuli; inhibiting platelet response to immunoreceptor tyrosine-based activation motif and G protein-coupled receptor signalling following stimulation with collagen, adenosine diphosphate, and thrombin, as well as enhancing integrin outside-in signalling. These dual, and opposing, roles suggest an important and complex role for PECAM-1 in orchestrating platelet response to vascular damage. Indeed, during thrombus formation, the influence of PECAM-1 on the multiple signalling pathways combines leading to a relatively large inhibitory effect on thrombus formation.     

Effects of inhibition of PAF, ICAM-1 and PECAM-1 on gut barrier failure caused by intestinal ischemia and reperfusion

BACKGROUND: The role of cell adhesion molecules and transmigration of PMNs through the endothelial barrier is probably essential in intestinal ischemia and reperfusion (I/R)-induced gut barrier dysfunction. Although cytokines are released in I/R, it is unclear whether cytokines directly increase permeability or if this phenomenon requires both expression of cell adhesion molecules and PMN adhesion-activation. Endothelial barrier dysfunction plays an important role in the pathogenesis of multiple organ dysfunction syndrome, inducing gut barrier failure, but the mechanisms are not fully understood. The purpose of this study was to evaluate the potential therapeutic value of inhibition of platelet activating factor (PAF), intercellular adhesion molecule-1 (ICAM-1), and platelet endothelial cell adhesion molecule-1 (PECAM-1) in gut barrier dysfunction induced by intestinal I/R. METHODS: A PAF antagonist (lexipafant, BB-882) and monoclonal antibodies against rat ICAM-1 (anti-ICAM-1-MAb) and PECAM-1 (anti-PECAM-1-MAb) were used in a model of gut barrier dysfunction caused by intestinal ischemia for 40 min and concomitant reperfusion for 12 h in the rat, and endothelial permeability, myeloperoxidase activity, interleukin-1beta and protease inhibitor levels were evaluated. RESULTS: The endothelial permeability and tissue leukocyte recruitment in the distal small intestine significantly increased in rats with I/R treated with saline. Proteolytic activity in plasma was evident by low levels of the three measured plasma protease inhibitors. These changes were, to different degrees, reduced by treatment with lexipafant, anti-ICAM-1-MAb, or anti-PECAM-1-MAb. Alterations in systemic levels of interleukin-1beta paralleled the changes found in gut barrier permeability and leukocyte trapping. CONCLUSIONS: Our results suggest that treatment with the PAF inhibitor lexipafant and monoclonal antibodies against ICAM-1 or, seemingly most efficient, PECAM-1 reduces the severity of I/R-associated intestinal dysfunction, associated with a decrease in systemic concentrations of IL-1beta local leukocyte recruitment, and partly restoring plasma protease inhibitor levels.     

CLEVER-1 mediates lymphocyte transmigration through vascular and lymphatic endothelium

Common lymphatic endothelial and vascular endothelial receptor-1 (CLEVER-1; also known as stabilin-1 or FEEL-1) is a large multifunctional glycoprotein implicated in scavenging, angiogenesis, and cell adhesion. Here we studied the function of human CLEVER-1 in leukocyte trafficking. Lymphatic vessels expressed CLEVER-1 constitutively in skin in vivo, whereas on vascular endothelium it appeared only upon inflammation. On isolated vascular endothelial cells, CLEVER-1 supported rolling and transmigration of peripheral blood mononuclear cells (PBMCs) under physiologically relevant laminar shear stress. Intriguingly, CLEVER-1 also mediated transmigration of leukocytes through cultured lymphatic endothelium under static conditions. Thus, synthesis of CLEVER-1 is differentially regulated on the 2 anatomically distinct vascular beds, and CLEVER-1 mediates the transmigration step of the leukocyte traffic in both of them. Notably, CLEVER-1 is the first adhesion molecule shown to be involved in the PBMC transmigration through the lymphatic arm of the immune system.     

PECAM-1 functions as a specific and potent inhibitor of mitochondrial-dependent apoptosis

Programmed cell death, or apoptosis, is a tightly regulated, naturally occurring process by which damaged or unwanted cells are removed. Dysregulated apoptosis has been implicated in a variety of pathophysiological conditions, including degenerative diseases, tissue remodeling, and tumorigenesis. The decision to live or die results from integration of numerous environmental signals transmitted by specific classes of cell surface receptors that bind hormones, growth factors, or components of the extracellular matrix. Here we show that platelet endothelial cell adhesion molecule-1 (PECAM-1), a homophilic-binding member of the immunoreceptor tyrosine-based inhibitory motif (ITIM) family of inhibitory receptors, functions prominently to inhibit apoptosis in naturally occurring vascular cells subjected to apoptotic stimuli. Murine endothelial cells and human T lymphocytes lacking PECAM-1 were found to be far more sensitive than their PECAM-1-expressing counterparts to multiple death signals that stimulate Bax, a multidomain, proapoptotic member of the Bcl-2 family that plays a central role in mitochondrial dysfunction-dependent apoptosis. In addition, PECAM-1 markedly suppressed Bax overexpression-induced cytochrome c release, caspase activation, and nuclear fragmentation. Amino acid substitutions within PECAM-1's extracellular homophilic binding domain, or within its cytoplasmic ITIM, completely abolished PECAM-1-mediated cytoprotection. Taken together, these data implicate PECAM-1 as a novel and potent suppressor of Bax-mediated apoptosis and suggest that members of the immunoglobulin gene (Ig) superfamily, like cell surface integrins, may also transmit survival signals into blood and vascular cells.     

Organization of the gene for human platelet/endothelial cell adhesion molecule-1 shows alternatively spliced isoforms and a functionally complex cytoplasmic domain

Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a cell-cell adhesion molecule that is expressed on circulating platelets, on leukocytes, and at the intercellular junctions of vascular endothelial cells and mediates the interactions of these cells during the process of transendothelial cell migration. The cDNA for PECAM-1 encodes an open reading frame of 738 amino acids (aa) that is organized into a 27- aa signal peptide, a 574-aa extracellular domain composed of 6 Ig homology units, and a relatively long cytoplasmic tail of 118 aa containing multiple sites for posttranslational modification and postreceptor signal transduction. To provide a molecular basis for the precise evaluation of the structure and function of this transmembrane glycoprotein, we have determined the organization of the human PECAM-1 gene. The PECAM-1 gene, which has been localized to human chromosome 17, is a single-copy gene of approximately 65 kb in length and is broken into 16 exons by introns ranging in size from 86 to greater than 12,000 bp in length. Typical of other members of the Ig superfamily, each of the extracellular Ig homology domains is encoded by a separate exon, consistent with PECAM-1 having arisen by gene duplication and exon shuffling of ancestral Ig superfamily genes. However, the cytoplasmic domain was found to be surprisingly complex, being encoded by seven short exons that may represent discrete functional entities. Alternative splicing of the cytoplasmic tail appears to generate multiple PECAM-1 isoforms that may regulate phosphorylation, cytoskeletal association, and affinity modulation of the mature protein. Finally, a processed pseudogene having 76% identity with PECAM- 1 cDNA was identified and localized to human chromosome 3. These findings should have important implications for structure/function analysis of PECAM-1 and its role in vascular adhesive interactions.     

Vasculogenesis,angiogenesis and the molecular organisation of endothelial junctions in the early human placenta

Vasculogenesis and angiogenesis are regulated by the capacity of endothelial cells to adhere to each other and form new tubes. The presence and role of junctional adhesion molecules during physiological vasculogenesis is unknown. Using ultrastructural and immunocytochemical approaches, we compared the junctional phenotype of developing vessels of the first-trimester human placenta with vessels in the last trimester; the latter include newly formed terminal capillaries and the quiescent vascular bed. First-trimester placental vessels contained the adherens junctional molecules, vascular endothelial cadherin and alpha- and beta-catenin but lacked plakoglobin, the component of fully differentiated adherens junctions. Furthermore, these vessels did not contain the transmembrane tight junctional molecules occludin and claudin-1 and -2. This profile reflects the phenotype of terminal capillaries but differs from large vessels of the full-term placenta. Electron microscopic studies revealed that endothelial tight junctions are present in the first-trimester placenta. Thus, occludin and claudin-1 appear to play no part in the formation of endothelial tight junctions, but are a later requirement. In the early placenta, the predominant growth factor appears to be vascular endothelial growth factor (VEGF), whilst at term, angiopoietin-1 was present in large vessels, with intense angiopoietin-2 immunofluorescence (and VEGF) located in terminal villous capillaries. Thus, endothelial junctions in the human placenta possess two distinct molecular phenotypes, i.e. stable or dynamic, dependent on maturity and plasticity. These distinct phenotypes may be influenced by the angiopoietins/VEGF present in the placenta.     

Neutrophil recruitment under shear flow: it's all about endothelial cell rings and gaps

Leukocyte recruitment to tissues and organs is an essential component of host defense. The molecular mechanisms controlling this process are complex and remain under active investigation. The combination of biochemical techniques and live cell imaging using in vivo and in vitro flow-model approaches have shed light on several aspects of neutrophil transmigration through the vascular endothelial lining of blood vessels. Here, we focus on the role of adhesion molecule signaling in endothelial cells and their downstream targets during the process of transendothelial migration at cell-cell borders (paracellular transmigration). An emerging model involves the leukocyte beta2 integrin engagement of endothelial cell ICAM-1, which triggers integrin-ICAM-1 clustering (rings) and stabilizes leukocyte adhesion at cell-cell junctions. This step recruits nonreceptor tyrosine kinases that phosphorylate key tyrosine residues in the cytoplasmic tail of VE-cadherin, which destabilizes its linkage to catenins and the actin cytoskeleton, triggering the transient opening of VE-cadherin homodimers to form a gap in the cell junction, through which the neutrophil transmigrates. Interestingly, the signaling events that lead to neutrophil transmigration occur independently of shear flow in vitro.     

Platelet Endothelial Cell Adhesion Molecule 1 (PECAM-1/CD31): A Multifunctional Vascular Cell Adhesion Molecule

PECAM-1/CD31 is a member of the immunoglobulin gene superfamily found on platelets, leukocytes, and endothelial cells, where it concentrates at cell-cell borders. It has been shown to both mediate cell-cell adhesion through homophilic and heterophilic interactions and to transduce intracellular signals that upregulate the function of integrins on leukocytes. Its cellular distribution and ability to mediate adhesive and signaling phenomena suggested that PECAM-1 was a multifunctional vascular cell adhesion molecule involved in leukocyte-endothelial and endothelial-endothelial interactions. These initial suggestions have been largely confirmed as recent studies have implicated PECAM-1 in the inflammatory process and in the formation of blood vessels. As our understanding of the molecular and functional properties of PECAM-1 grows, new insights will be gained that may have therapeutic implications for cardiovascular development and disease. (Trends Cardiovasc Med 1997;7:203-210). ? 1997, Elsevier Science Inc.     

Extracellular matrix mediates a molecular balance between vascular morphogenesis and regression

PURPOSE OF REVIEW: We discuss very recent studies that address the critical role of extracellular matrix in controlling the balance between vascular morphogenesis and regression. Much of this work suggests that a balance mechanism exists for controlling the extent of tissue vascularization involving downstream signaling events regulating endothelial cell behaviors in relation to their interactions with extracellular matrix molecules. RECENT FINDINGS: Endothelial gene expression changes and signaling lead to events that not only stimulate vascular morphogenesis but also suppress mechanisms mediated through pro-regression factors such as Rho kinase. At the same time, vascular networks are susceptible to regression mediated by factors such as matrix metalloproteinase-1, matrix metalloproteinase-10, thrombospondin-1, extracellular matrix matricryptic fragments and angiopoietin-2. Pericyte recruitment to such vascular tubes can prevent regression events by delivering molecules such as tissue inhibitor of metalloproteinase-3 and angiopoietin-1 that promote vascular stabilization by decreasing tube susceptibility to these regression stimuli. SUMMARY: Extracellular matrix-derived signals lead to critical morphologic changes mediated through cytoskeletal rearrangements that control the shape, function and signaling events in endothelial cell-lined vessels regulating tube formation, remodeling, stabilization and regression. These signals control both vascular morphogenic and regression events, and thus a molecular balance exists to control the extent and function of vascular tube networks within tissues.