Endothelial permeability and VE-cadherin

The endothelium forms a selective semi-permeable barrier controlling bidirectional transfer between blood vessel and irrigated tissues. This crucial function relies on the dynamic architecture of endothelial cell–cell junctions, and in particular, VE-cadherin-mediated contacts. VE-cadherin indeed chiefly organizes the opening and closing of the endothelial barrier, and is central in permeability changes. In this review, the way VE-cadherin-based contacts are formed and maintained is first presented, including molecular traits of its expression, partners, and signaling. In a second part, the mechanisms by which VE-cadherin adhesion can be disrupted, leading to cell–cell junction weakening and endothelial permeability increase, are described. Overall, the molecular basis for VE-cadherin control of the endothelial barrier function is of high interest for biomedical research, as vascular leakage is observed in many pathological conditions and human diseases.     

Endothelial permeability and VE-cadherin

The endothelium forms a selective semi-permeable barrier controlling bidirectional transfer between blood vessel and irrigated tissues. This crucial function relies on the dynamic architecture of endothelial cell–cell junctions, and in particular, VE-cadherin-mediated contacts. VE-cadherin indeed chiefly organizes the opening and closing of the endothelial barrier, and is central in permeability changes. In this review, the way VE-cadherin-based contacts are formed and maintained is first presented, including molecular traits of its expression, partners, and signaling. In a second part, the mechanisms by which VE-cadherin adhesion can be disrupted, leading to cell–cell junction weakening and endothelial permeability increase, are described. Overall, the molecular basis for VE-cadherin control of the endothelial barrier function is of high interest for biomedical research, as vascular leakage is observed in many pathological conditions and human diseases.     

Endothelial permeability and VE-cadherin: A wacky comradeship

The endothelium forms a selective semi-permeable barrier controlling bidirectional transfer between blood vessel and irrigated tissues. This crucial function relies on the dynamic architecture of endothelial cell–cell junctions, and in particular, VE-cadherin-mediated contacts. VE-cadherin indeed chiefly organizes the opening and closing of the endothelial barrier, and is central in permeability changes. In this review, the way VE-cadherin-based contacts are formed and maintained is first presented, including molecular traits of its expression, partners, and signaling. In a second part, the mechanisms by which VE-cadherin adhesion can be disrupted, leading to cell–cell junction weakening and endothelial permeability increase, are described. Overall, the molecular basis for VE-cadherin control of the endothelial barrier function is of high interest for biomedical research, as vascular leakage is observed in many pathological conditions and human diseases.     

Endothelial permeability and VE-cadherin: A wacky comradeship

The endothelium forms a selective semi-permeable barrier controlling bidirectional transfer between blood vessel and irrigated tissues. This crucial function relies on the dynamic architecture of endothelial cell–cell junctions, and in particular, VE-cadherin-mediated contacts. VE-cadherin indeed chiefly organizes the opening and closing of the endothelial barrier, and is central in permeability changes. In this review, the way VE-cadherin-based contacts are formed and maintained is first presented, including molecular traits of its expression, partners, and signaling. In a second part, the mechanisms by which VE-cadherin adhesion can be disrupted, leading to cell–cell junction weakening and endothelial permeability increase, are described. Overall, the molecular basis for VE-cadherin control of the endothelial barrier function is of high interest for biomedical research, as vascular leakage is observed in many pathological conditions and human diseases.     

Molecular events during leukocyte diapedesis

The recruitment of leukocytes from the circulation into tissues requires leukocyte migration through the vascular endothelium. The mechanisms by which leukocytes attach and firmly adhere to the endothelial cell surface have been studied in detail. However, much less is known about the last step in this process, the diapedesis of leukocytes through the vascular endothelium. This minireview focuses on the interactions between leukocyte and endothelial cell adhesion molecules that are important during leukocyte extravasation. In the past few years a series of endothelial cell surface and adhesion molecules have been identified that are located at endothelial cell contacts and found to participate in leukocyte diapedesis. These junctional cell adhesion molecules are believed to have an active role in controlling the opening and closure of endothelial cell contacts to allow the passage of leukocytes between adjacent endothelial cells. Alternatively, leukocytes can cross the endothelium at nonjunctional locations, with leukocytes migrating through a single endothelial cell. Further work is clearly needed to understand, in greater detail, the molecular mechanisms that allow leukocytes to cross the endothelium via either the paracellular or the transcellular pathway.     

Cell-cell signaling by direct contact increases cell proliferation via a PI3K-dependent signal

We report a novel mechanism of cellular growth control. Increasing the density of endothelial or smooth muscle cells in culture increased cell-cell contact and decreased cell spreading, leading to growth arrest. Using a new method to independently control cell-cell contact and cell spreading, we found that introducing cell-cell contact positively regulates proliferation, but that contact-mediated proliferation can be masked by changes in cell spreading: Round cells with many contacts proliferated less than spread cells with none. Physically blocking cell-cell contact or inhibiting PI3K signaling abrogated cell-cell induced proliferation, but inhibiting diffusible paracrine signaling did not. Thus, direct cell-cell contact induces proliferation in these cells.     

ADAM15 is an adherens junction molecule whose surface expression can be driven by VE-cadherin

ADAM15 belongs to the family of proteins containing disintegrin and metalloprotease domains (ADAM) that have been implicated in cell adhesion via integrin binding and shedding of cell surface molecules. Here we provide the first report on the localization of an ADAM in adherens junctions. We show that ADAM15 colocalizes with a cell adhesion molecule, vascular endothelial (VE)-cadherin, which mediates endothelial cell adherens junction formation. In contrast, the distribution of ADAM15 correlates poorly with the localization in cell contacts of one of its proposed ligands, the beta1-integrin. Furthermore, ADAM15 accumulation in cell-cell contacts is preceded by VE-cadherin-mediated adherens junction formation. To investigate the dependence of ADAM15 surface expression on adherens junction formation, we coexpressed VE-cadherin with ADAM15 and an ADAM15 green fluorescence protein (GFP) fusion protein in Chinese hamster ovary cells. VE-cadherin coexpression results in the translocation of ADAM15-GFP to the cell periphery. Analysis of cell surface levels of ADAM15 and ADAM15-GFP, with or without VE-cadherin coexpression, clearly demonstrates that VE-cadherin can drive surface expression of ADAM15. Our data suggest that ADAM15 may be a novel component of adherens junctions and thus could play a role in endothelial functions that are mediated by these cell contacts.     

Cell-cell junctions of dermal microvascular endothelial cells contain tight and adherens junction proteins in spatial proximity

Endothelial cell-cell contacts control the vascular permeability, thereby regulating the flow of solutes, macromolecules, and leukocytes between blood vessels and interstitial space. Because of specific needs, the endothelial permeability differs significantly between the tight blood-brain barrier endothelium and the more permeable endothelial lining of the non-brain microvasculature. Most likely, such differences are due to a differing architecture of the respective interendothelial cell contacts. However, while the molecules and junctional complexes of macrovascular endothelial cells and the blood-brain barrier endothelium are fairly well characterized, much less is known about the organization of intercellular contacts of microvascular endothelium. Toward this end, we developed a combined cross-linking and immunoprecipitation protocol which enabled us to map nearest neighbor interactions of junctional proteins in the human dermal microvascular endothelial cell line HMEC-1. We show that proteins typically located in tight or adherens junctions of epithelial cells are in the proximity in HMEC-1 cells. This contrasts with the separation of the different types of junctions observed in polarized epithelial cells and "tight" endothelial layers of the blood-brain barrier and argues for a need of the specific junctional contacts in microvascular endothelium possibly required to support an efficient transendothelial migration of leukocytes.     

Monoclonal antibody to CD9 inhibits platelet-induced human endothelial cell proliferation

Platelets are anucleate cytoplasmic fragments derived from bone marrow megakaryocytes, and endothelial cells constitute the barrier between bloodstream and adjacent tissues. Although platelets are thought to regulate the biological functions of endothelial cells, the molecular mechanisms involved are poorly understood. With human umbilical vein endothelial cells and freshly isolated platelets, we established an in vitro model of platelet-induced endothelial cell proliferation. Platelets stimulated endothelial cell proliferation in a dose-dependent manner and transwell experiments with semi-permeable membranes suggested that direct cell-to-cell contacts were required. We developed mAbs against platelets and selected a mAb that blocks their proliferative effect. We purified the antigen by immunoprecipitation and identified it by Q-TOF MS analysis as the tetraspanin CD9. Since both platelets and endothelial cells expressed CD9 strongly on their surfaces we carried out a pre-treatment experiment that showed that CD9 molecules on the endothelial cells participate in the mitogenic effect of the platelets. The inhibitory effect of our mAb was comparable to that of a well-known functional anti-CD9 mAb. These results suggest that the tetraspanin CD9 plays an important role in endothelial regeneration.     

A coronary artery disease-associated gene product, JCAD/KIAA1462, is a novel component of endothelial cell-cell junctions

Cell–cell junctions play crucial roles in the organization and function of epithelial and endothelial cellular sheets. Here, we have identified the protein product for KIAA1462 gene, whose single nucleotide polymorphisms (SNPs) have recently reported to be associated with coronary artery disease, as a novel component of cell–cell junctions. We propose the name of KIAA1462 protein junctional protein associated with coronary artery disease (JCAD). JCAD is a ∼145 kDa protein without any known domains but contains a proline-rich region. Immunolocalization studies revealed that JCAD is specifically localized at cell–cell junctions in endothelial cells but not in epithelial cells. The accumulation of JCAD at cell–cell junctions in cultured endothelial cells was impaired by RNAi-mediated suppression of VE-cadherin expression. In cell adhesion-deficient mouse L fibroblasts, JCAD was recruited to cell–cell contacts when cadherin-mediated cell–cell adhesion was induced. These results indicate that JCAD is a component of VE-cadherin-based cell–cell junctions in endothelial cells. This study also suggests the implication of endothelial cell–cell adhesion in coronary artery disease.     

Bcl‐2‐associated athanogene 5 overexpression attenuates catecholamine‐induced vascular endothelial cell apoptosis

Bcl‐2 associated athanogene 5 (Bag5) is a novel endoplasmic reticulum (ER) regulator. However, its role in catecholamine‐induced endothelial cells damage has not been fully understood. In our study, catecholamine was used to mimic hypertension‐related endothelial cell damage. Then, western blots, enzyme‐linked immunosorbent assay, immunofluorescence, quantitative polymerase chain reaction and pathway analysis were conducted to analyze the role of Bag5 in endothelial cell damage in response to catecholamine. Our results indicated that the endothelial cell viability was impaired by catecholamine. Interestingly, Bag5 overexpression significantly reversed endothelial cell viability. Mechanistically, Bag5 overexpression inhibited ER stress, attenuated oxidative stress and repressed inflammation in catecholamine‐treated endothelial cells. These beneficial effects finally contributed to endothelial cell survival under catecholamine treatment. Pathway analysis demonstrated that Bag5 was under the control of the mitogen‐activated protein kinase (MAPK)–extracellular‐signal‐regulated kinase (ERK) signaling pathway. Reactivation of the MAPK–ERK pathway could upregulate Bag5 expression and thus promote endothelial cell survival through inhibiting oxidative stress, ER stress, and inflammation. Altogether, our results illustrate that Bag5 overexpression sustains endothelial cell survival in response to catecholamine treatment. This finding identifies Bag5 downregulation and the inactivated MAPK–ERK pathway as potential mechanisms underlying catecholamine‐induced endothelial cell damage.     

Sphingosine 1-phosphate-induced endothelial cell migration requires the expression of EDG-1 and EDG-3 receptors and Rho-dependent activation of alpha vbeta3- and beta1-containing integrins

Sphingosine 1-phosphate (SPP), a platelet-derived bioactive lysophospholipid, is a regulator of angiogenesis. However, molecular mechanisms involved in SPP-induced angiogenic responses are not fully defined. Here we report the molecular mechanisms involved in SPP-induced human umbilical vein endothelial cell (HUVEC) adhesion and migration. SPP-induced HUVEC migration is potently inhibited by antisense phosphothioate oligonucleotides against EDG-1 as well as EDG-3 receptors. In addition, C3 exotoxin blocked SPP-induced cell attachment, spreading and migration on fibronectin-, vitronectin- and Matrigel-coated surfaces, suggesting that endothelial differentiation gene receptor signaling via the Rho pathway is critical for SPP-induced cell migration. Indeed, SPP induced Rho activation in an adherence-independent manner, whereas Rac activation was dispensible for cell attachment and focal contact formation. Interestingly, both EDG-1 and -3 receptors were required for Rho activation. Since integrins are critical for cell adhesion, migration, and angiogenesis, we examined the effects of blocking antibodies against alpha(v)beta(3), beta(1), or beta(3) integrins. SPP induced Rho-dependent integrin clustering into focal contact sites, which was essential for cell adhesion, spreading and migration. Blockage of alpha(v)beta(3)- or beta(1)-containing integrins inhibited SPP-induced HUVEC migration. Together our results suggest that endothelial differentiation gene receptor-mediated Rho signaling is required for the activation of integrin alpha(v)beta(3) as well as beta(1)-containing integrins, leading to the formation of initial focal contacts and endothelial cell migration.     

Angiopoietins assemble distinct Tie2 signalling complexes in endothelial cell-cell and cell-matrix contacts

The receptor tyrosine kinase Tie2, and its activating ligand Angiopoietin-1 (Ang1), are required for vascular remodelling and vessel integrity, whereas Ang2 may counteract these functions. However, it is not known how Tie2 transduces these different signals. Here, we show that Ang1 induces unique Tie2 complexes in mobile and confluent endothelial cells. Matrix-bound Ang1 induced cell adhesion, motility and Tie2 activation in cell-matrix contacts that became translocated to the trailing edge in migrating endothelial cells. In contrast, in contacting cells Ang1 induced Tie2 translocation to cell-cell contacts and the formation of homotypic Tie2-Tie2 trans-associated complexes that included the vascular endothelial phosphotyrosine phosphatase, leading to inhibition of paracellular permeability. Distinct signalling proteins were preferentially activated by Tie2 in the cell-matrix and cell-cell contacts, where Ang2 inhibited Ang1-induced Tie2 activation. This novel type of cellular microenvironment-dependent receptor tyrosine kinase activation may explain some of the effects of angiopoietins in angiogenesis and vessel stabilization.     

Mechanical interactions between dendritic cells and T cells correlate with T cell responsiveness

Ag recognition is achieved through the communication across intercellular contacts between T cells and APCs such as dendritic cells (DC). Despite remarkable progress in delineating detailed molecular components at the intercellular contacts, little is known about the functional roles of physical cross-junctional adhesion between T and DC in shaping T cell responses. In addition, the mechanisms underlying sensitivity and specificity of Ag discrimination by T cells at intercellular contacts remain to be elucidated. In this study, we use single-cell force spectroscopy to probe the mechanical interactions between DC and T cells in response to stimulation with a panel of altered peptide ligands. The results show that intercellular interactions of DC-T cell conjugates exhibited different ranges of interaction forces in peptide-dependent manners that match the ability of the peptides to activate T cells. Elevated calcium mobilization and IL-2 secretion by T cells were only promoted in response to antigenic peptides that induce strong interaction forces, suggesting that mechanically stable DC-T cell contacts are crucial for driving T cell activation. Strong interactions were not solely dependent on cell-surface molecules such as TCRs and the adhesion molecule LFA-1, but were also controlled by cytoskeletal dynamics and the integrity of membrane lipid rafts. These data provide novel mechanical insights into the effect of Ag affinity on intercellular contacts that align with T cell responsiveness.     

An endostatin-derived peptide interacts with integrins and regulates actin cytoskeleton and migration of endothelial cells

Endostatin, the C-terminal fragment of collagen XVIII, is a potent inhibitor of angiogenesis and endothelial cell migration. To define its critical cell interaction domains we used endostatin-derived synthetic peptides containing surface-exposed sequences. We observed that, when immobilized, an arginine-rich peptide of 11 amino acids from its N terminus efficiently promoted endothelial cell adhesion through beta(1) integrin- and heparin-dependent mechanisms. In addition, the peptide induced the formation of membrane ruffles and focal contacts. In the soluble form, the peptide inhibited basic fibroblast growth factor-induced directional migration and tubular morphogenesis of microvascular endothelial cells. Accordingly, the peptide induced the loss of focal adhesions and actin stress fibers in these cells. Substitution of the arginine residues with alanines resulted in the loss of these properties. In the current study we describe a putative integrin-binding sequence with anti-migratory activity within endostatin.     

The intermediate filament protein vimentin binds specifically to a recombinant integrin alpha2/beta1 cytoplasmic tail complex and co-localizes with native alpha2/beta1 in endothelial cell focal adhesions

Integrin receptors are crucial players in cell adhesion and migration. Identification and characterization of cellular proteins that interact with their short alpha and beta cytoplasmic tails will help to elucidate the molecular mechanisms by which integrins mediate bi-directional signaling across the plasma membrane. Integrin alpha2beta1 is a major collagen receptor but to date, only few proteins have been shown to interact with the alpha2 cytoplasmic tail or with the alpha2beta1 complex. In order to identify novel binding partners of a alpha2beta1cytoplasmic domain complex, we have generated recombinant GST-fusion proteins, incorporating the leucine zipper heterodimerization cassettes of Jun and Fos. To ascertain proper functionality of the recombinant proteins, interaction with natural binding partners was tested. GST-alpha2 and GST-Jun alpha2 bound His-tagged calreticulin while GST-beta1 and GST-Fos beta1 proteins bound talin. In screening assays for novel binding partners, the immobilized GST-Jun alpha2/GST-Fos beta1 heterodimeric complex, but not the single subunits, interacted specifically with endothelial cell-derived vimentin. Vimentin, an abundant intermediate filament protein, has previously been shown to co-localize with alphavbeta3-positive focal contacts. Here, we provide evidence that this interaction also occurs with alpha2beta1-enriched focal adhesions and we further show that this association is lost after prolonged adhesion of endothelial cells to collagen.