Endothelial Cell Contacts Research Articles

The changing cellular environments of hematopoiesis in human development in utero

The hematopoietic system is indispensable from the earliest stages of development and adapts to the rapidly changing anatomy of the embryo and fetus; this takes place in such different anatomic locations as the yolk sac blood island, hepatic parenchyme, aorta-gonads-mesonephros paravascular mesenchyme, and bone marrow primary logette. We herein summarize our investigation of these serial blood-forming events in the human embryo and fetus. The access to early stages of human development, availability of a large panoply of molecular markers for human blood cell lineages, and recent development of robust assays for the earliest human hematopoietic stem cells have allowed us to gain relatively clear insight into the developmental sequence that underlies the ontogeny of human blood cells. Conversely, the control exerted by these diverse cellular environments on the emergence of human hematopoietic cells remains elusive, as is the case in animal models. We nonetheless present preliminary attempts to decipher the structure and molecular characteristics of the distinct cellular "niches" in which blood cells are produced during human gestation.

The Conserved Immunoglobulin Domain Controls the Subcellular Localization of the Homophilic Adhesion Receptor Protein-tyrosine Phosphatase μ

The receptor protein-tyrosine phosphatase mu (PTPmu) is a homophilic adhesion protein thought to regulate cell-cell adhesion in the vascular endothelium through dephosphorylation of cell junction proteins. In subconfluent cell cultures, PTPmu resides in an intracellular membrane pool; however, as culture density increases and cell contacts form, the phosphatase localizes to sites of cell-cell contact, and its expression level increases. These characteristics of PTPmu, which are consistent with a role in cell-cell adhesion, suggest that control of subcellular localization is an important mechanism to regulate the function of this phosphatase. To gain a better understanding of how PTPmu is regulated, we examined the importance of the conserved immunoglobulin domain, containing the homophilic binding site, in control of the localization of the enzyme. Deletion of the immunoglobulin domain impaired localization of PTPmu to the cell-cell contacts in endothelial and epithelial cells. In addition, deletion of the immunoglobulin domain affected the distribution of PTPmu in subconfluent endothelial cells when homophilic binding to another PTPmu molecule on an apposing cell was not possible, resulting in an accumulation of the mutant phosphatase at the cell surface with a concentration at the cell periphery in the region occupied by focal adhesions. This aberrant localization correlated with reduced survival and alterations in normal focal adhesion and cytoskeleton morphology. This study therefore illustrates the critical role of the immunoglobulin domain in regulation of the localization of PTPmu and the importance of such control for the maintenance of normal cell physiology.

The Expression of Microfilament-Associated Cell-Cell Contacts in Brain Endothelial Cells Is Modified by IFN-β1a (Rebif®)

In multiple sclerosis (MS), a crucial step in the induction phase of the inflammatory process in the central nervous system (CNS) is the disruption of the endothelial blood-brain barrier (BBB). Its permeability depends on the expression of intercellular adhesion molecules, such as vinculin and N-cadherin in endothelial cells. Interferon-gamma (IFN-gamma), as a proinflammatory cytokine, decreases the expression of both adhesion molecules in epithelial and astrocytic cells, whereas IFN-beta1a, an established treatment for MS, increases the expression of N-cadherin and vinculin in astrocytic cells and is postulated to preserve endothelial cell barrier function and to inhibit transendothelial migration of activated leukocytes. We analyzed the expression of N-cadherin and vinculin in a murine brain endothelial cell line by immunofluorescence staining and Western blot to study the presumed reversal effects of IFN-beta1a (Rebif, Serono Pharma, Unterschleissheim, Germany) and IFN-gamma on the formation of intercellular contacts. Vinculin and N-cadherin expression in brain endothelial cells was decreased after treatment with IFN-gamma, whereas stimulation with IFN-beta1a caused increased expression of both adhesion molecules. Combined treatment with both IFNs did not affect vinculin and N-cadherin expression. These data suggest that IFN-gamma contributes to BBB disruption by decreasing and IFN-beta1a restores the BBB by an upregulation of vinculin and N-cadherin expression in brain endothelial cells. This action of IFN-beta1a may contribute to its beneficial effects in MS therapy.

The Expression of Microfilament-Associated Cell-Cell Contacts in Brain Endothelial Cells Is Modified by IFN-<I>β</I>1a (Rebif<SUP>®</SUP>)

The Expression of Microfilament-Associated Cell-Cell Contacts in Brain Endothelial Cells Is Modified by IFN-<I>β</I>1a (Rebif<SUP>®</SUP>)

Mouse CD99 participates in T-cell recruitment into inflamed skin

AbstractHuman CD99 is a small highly O-glycosylated cell-surface protein expressed on most leukocytes. It was recently found to be expressed at endothelial cell contacts and to participate in the transendothelial migration (TEM) of monocytes in vitro. In order to analyze the physiologic relevance of CD99 in vivo we searched for the mouse homolog. We cloned a mouse cDNA coding for a protein 45% identical in its sequence with human CD99. Based on the cDNA, we generated antibodies against this mouse homolog of CD99, which detected the antigen on most leukocytes, on endothelia of various tissues, and at cell contacts of cultured endothelial cells. Cell aggregation of CD99-transfected Chinese hamster ovary (CHO) cells was completely blocked by anti-CD99 antibodies. The same antibodies inhibited TEM of lymphocytes in vitro, independent of whether T cells or endothelial cells were preincubated with antibodies. In a cutaneous delayed-type hypersensitivity (DTH) reaction, anti-CD99 antibodies inhibited the recruitment of in vivo–activated T cells into inflamed skin as well as edema formation. We conclude that mouse CD99 participates in the TEM of lymphocytes and in their recruitment to inflamed skin in vivo. This establishes CD99 as a valid target for interference with cutaneous inflammatory processes.

Colocalization of eNOS and the catalytic subunit of PKA in endothelial cell junctions: a clue for regulated NO production.

Localization and coordinate phosphorylation/dephosphorylation of endothelial nitric oxide synthase (eNOS) are critical determinants for the basal and stimulated production of nitric oxide. Several phosphorylation sites in eNOS have been identified as targets of the cAMP-dependent protein kinase A (PKA). Basal eNOS activity is also regulated by interaction with caveolin-1, the major coat protein of caveolae. In the present study we have examined in rat aorta endothelium the subcellular steady-state distribution of eNOS, the catalytic subunit of PKA (PKA-c), and caveolin-1. Basal eNOS expression was found in two distinct locations, the endothelial cell surface and the Golgi complex. Cell surface eNOS was equally distributed over caveolar and non-caveolar membranes but was 2.5-fold enriched on luminal lamellipodia located at endothelial cell contacts. PKA-c colocalized with eNOS in the lamellipodia, whereas caveolin-1 was absent from these membrane domains. PKA-c was also found associated with cell surface caveolae and with tubulovesicular membranes of Golgi complex and endosomes. The topological proximity of eNOS with the catalytic subunit of PKA in restricted intracellular locations may provide mechanisms for differential PKA-mediated eNOS regulation.

Endothelial adhesion molecule ESAM binds directly to the multidomain adaptor MAGI-1 and recruits it to cell contacts

Abstract Endothelial cell-selective adhesion molecule (ESAM) is an immunoglobulin-like transmembrane protein associated with endothelial tight junctions (TJ). Based on a yeast two-hybrid screen, we have identified the membrane-associated guanylate kinase protein MAGI-1 as an intracellular binding partner of ESAM. MAGI-1 is a multidomain adaptor protein, which binds to transmembrane, cytoskeletal, and signaling molecules, and has been localized to tight junctions in epithelial cells. MAGI-1 associates with the very C-terminal sequence of ESAM most likely through a PDZ domain-mediated interaction. The direct interaction between ESAM and MAGI-1 was confirmed by pull-down experiments. The two proteins formed stable complexes in transfected Chinese hamster ovary (CHO) cells, which could be immunoisolated. We found MAGI-1 to be associated with cell–cell contacts in human umbilical vein endothelial cells (HUVECs) and in mouse endothelium, where it colocalizes with ESAM. In CHO cells, recruitment of MAGI-1 to cell contacts required the presence of ESAM. Hence, ESAM may be involved in anchoring MAGI-1 at endothelial tight junctions.

Endothelial adhesion molecule ESAM binds directly to the multidomain adaptor MAGI-1 and recruits it to cell contacts

Endothelial cell-selective adhesion molecule (ESAM) is an immunoglobulin-like transmembrane protein associated with endothelial tight junctions (TJ). Based on a yeast two-hybrid screen, we have identified the membrane-associated guanylate kinase protein MAGI-1 as an intracellular binding partner of ESAM. MAGI-1 is a multidomain adaptor protein, which binds to transmembrane, cytoskeletal, and signaling molecules, and has been localized to tight junctions in epithelial cells. MAGI-1 associates with the very C-terminal sequence of ESAM most likely through a PDZ domain-mediated interaction. The direct interaction between ESAM and MAGI-1 was confirmed by pull-down experiments. The two proteins formed stable complexes in transfected Chinese hamster ovary (CHO) cells, which could be immunoisolated. We found MAGI-1 to be associated with cell–cell contacts in human umbilical vein endothelial cells (HUVECs) and in mouse endothelium, where it colocalizes with ESAM. In CHO cells, recruitment of MAGI-1 to cell contacts required the presence of ESAM. Hence, ESAM may be involved in anchoring MAGI-1 at endothelial tight junctions.

Functional consequences of immune cell adhesion to endothelial cells.

Research regarding the interactions between the endothelium and immune cells has undergone a significant expansion during the past decade. Major shifts of emphasis have been the norm, from the production of a detail catalog of the cell surface receptors and counter-receptors acting at the interface between the vascular endothelium and circulating cells to a more mechanistic account of leukocyte/endothelium interactions. The past five years has seen new, groundbreaking developments in the field, with exiting studies aimed at understanding the functional consequences of the direct contact of endothelial cells and leukocytes. Based on early work to be discussed below, new data on local chemokine production and cell-to-cell contacts, attempt to clarify the physiopathological significance of these events. The exceptional anatomical arrangement of endothelial cells insures a permanent contact of the endothelium with leukocytes, an event likely to result in cellular signals originating from direct cell contact or through the action of soluble factors produced by endothelial cells or immune cells. As we will discuss, current evidence supports the idea that endothelial cells present at vascular endothelium as well as at specialized high endothelial venules, play not only a critical role in the homing and recruitment of immune cells but that it can also influence the outcome of the immune response. Additionally, new evidence clearly corroborates the idea that B and T lymphocytes as well as NK cells can modulate endothelial cell function.

The vimentin cytoskeleton regulates focal contact size and adhesion of endothelial cells subjected to shear stress.

Recently, we reported that vimentin-type intermediate filaments, in addition to microfilaments, associate with alphavbeta3 integrin-positive focal contacts in endothelial cells. To gain insight into intermediate filament-focal contact interaction, we induced expression of yellow fluorescent protein (YFP)-integrin beta3 and cyan fluorescent protein (CFP)-vimentin protein in endothelial cells. At least 50% of the YFP-beta3 integrin-labeled focal contacts associated with CFP-labeled vimentin intermediate filaments in live cells. Moreover, focal contacts and intermediate filaments moved in concert in the plane of the membrane and assembling focal contacts were sites of vimentin filament assembly. When endothelial cells were subjected to flow, large focal contacts assembled and associated with thick vimentin bundles. These large focal contacts showed minimal dynamic activity. Cells in which vimentin expression had been inhibited by RNA interference assembled smaller than normal focal contacts. More dramatically, such cells showed decreased adhesion to the substratum. These data provide evidence that the vimentin cytoskeleton regulates focal contact size and helps stabilize cell-matrix adhesions in endothelial cells.

Lumen formation: in vivo versus in vitro observations.

Lumen formation must accompany the de novo growth of blood vessels during embryological development, the production of new vessels (vasculogenesis), and the expansion or remodeling of the microcirculation in differentiated tissue (angiogenesis). The debate over lumen origin centers on whether this is an intracellular or intercellular phenomenon, entailing vesicle accretion or loss of endothelial cell (EC) contact, and whether this represents an intrinsic property of ECs or relies on extrinsic signals. In addition, recent in vivo data suggest that a third mechanism, that of longitudinal division, may be used to expand existing capillary networks. Importantly, more than one mechanism of lumen formation may be found in response to a given angiogenic signal. Tubule formation by ECs in a matrix is an increasingly popular form of in vitro angiogenesis assay, and it may offer insights into the mechanisms involved during growth in embryos or under pathological conditions in adults. Crucial to the validity of in vitro preparations is the extent to which tubule assembly and lumen formation mirrors that observed in vivo, although these data cannot elucidate the controls operative during adaptive remodeling of the vascular bed. Similar structures may be observed in vivo and in vitro, and may represent the situation found during angiogenesis and vasculogenesis, respectively. Lumen formation during angiogenesis, and tubule formation during EC culture, require the existence of cell polarity. As tubule formation is not a unique property of ECs, how this is developed is a key area where in vitro studies may extend our understanding of EC biology.

Lumen FormationIn VivoVersusIn VitroObservations

Lumen formation must accompany the de novogrowth of blood vessels during embryological development, the production of new vessels (vasculogenesis), and the expansion or remodeling of the microcirculation in differentiated tissue (angiogenesis). The debate over lumen origin centers on whether this is an intracellular or intercellular phenomenon, entailing vesicle accretion or loss of endothelial cell (EC) contact, and whether this represents an intrinsic property of ECs or relies on extrinsic signals. In addition, recent in vivodata suggest that a third mechanism, that of longitudinal division, may be used to expand existing capillary networks. Importantly, more than one mechanism of lumen formation may be found in response to a given angiogenic signal. Tubule formation by ECs in a matrix is an increasingly popular form of in vitroangiogenesis assay, and it may offer insights into the mechanisms involved during growth in embryos or under pathological conditions in adults. Crucial to the validity of in vitr...

Corneal endothelial cell density and pachymetry measured by contact and noncontact specular microscopy

Purpose To determine the endothelial cell density and thickness of normal human and postkeratoplasty corneas with contact specular microscopy and to compare these measurements with those obtained by noncontact specular microscopy. Setting Department of Ophthalmology, University of Erlangen-Nürnberg, Erlangen, Germany. Methods The central corneal endothelial cell density and thickness were determined in 65 healthy eyes of 39 patients with a mean age of 71 years ± 12 (SD) and in 50 corneal grafts of 41 patients with a mean age 53 ± 17 years using noncontact (Topcon SP-2000P, Topcon Corp.) and contact (EM-1000, Tomey) specular microscopes. Appropriate conversion factors were used for accurate cell count comparison. Results The mean cell count of the normal corneas was 2445 ± 425 cells/mm 2 measured by noncontact specular microscopy and 2471 ± 393 cells/mm 2 measured by contact specular microscopy ( P = .70). After penetrating keratoplasty, the mean cell density was 1610 ± 499 cells/mm 2 and 1584 ± 469 cells/mm 2, respectively ( P = .88). Significantly lower thickness was measured with the noncontact specular microscope than by contact pachymetry in normal eyes (543 ± 46 μm and 642 ± 42 μm, respectively) and postkeratoplasty eyes (538 ± 61 μm and 627 ± 48 μm, respectively) ( P < .0001). Conclusions To determine endothelial cell density, contact and noncontact specular microscopy may be used interchangeably. However, for the combined measurement of endothelial cell density and pachymetry, the use of the same specular microscope is recommended for long-term patient follow-up.

Tightening of Endothelial Cell Contacts: A Physiologic Response to Cocultures with Smooth-Muscle-Like 10T1/2 Cells

Tightening of endothelial cell-to-cell contacts is an important event at the end of angiogenesis in order to achieve controlled transfer of solutes between the blood stream and solid tissues. We found that tightening of endothelial cell-to-cell contacts and the formation of a permeability barrier can be induced in vitro by dibutyryl cAMP and hydrocortisone. This process is accompanied by increased junctional localization and cytoskeletal association of the adherens junctional plakoglobin and the tight junction associated proteins ZO-1, ZO-2, and occludin. Based on these findings, we proceeded to investigate whether smooth-muscle-like mesenchymal cells would influence endothelial junctional differentiation. For this purpose, human umbilical chord vein endothelial cells and murine smooth-muscle-like 10T1/2 cells were cocultivated and compared with their respective monocultures. Immunofluorescence on cells and Western blot analyses were performed for marker proteins of adherens and tight junctions. Functional permeability assays were performed for the tracer molecule biotin-dextran. The results indicated that 10T1/2 cells induced the tightening of endothelial cell-to-cell contacts. Plakoglobin, ZO-1, ZO-2, and occludin showed increased junctional localization when 10T1/2 cells were present. Cocultures also displayed a significantly higher permeability barrier for the tracer molecule biotin-dextran. In conclusion, mural cells such as smooth muscle cells and pericytes may be important for stabilizing endothelial cell-to-cell contacts and may influence vessel-type specific differences of the endothelial phenotype.

A Transmembrane Tight Junction Protein Selectively Expressed on Endothelial Cells and Platelets

Searching for cell surface proteins expressed at interendothelial cell contacts, we have raised monoclonal antibodies against intact mouse endothelial cells. We obtained two monoclonal antibodies, 1G8 and 4C10, that stain endothelial cell contacts and recognize a protein of 55 kDa. Purification and identification by mass spectrometry of this protein revealed that it contains two extracellular Ig domains, reminiscent of the JAM family, but a much longer 120-amino acid cytoplasmic domain. The antigen is exclusively expressed on endothelial cells of various organs as was analyzed by immunohistochemistry. Immunogold labeling of ultrathin sections of brain as well as skeletal muscle revealed that the antigen strictly colocalizes in capillaries with the tight junction markers occludin, claudin-5, and ZO-1. Upon transfection into MDCK cells, the antigen was restricted to the most apical tip of the lateral cell surface, where it colocalized with ZO-1 but not with beta-catenin. In contrast to JAM-1, however, the 1G8 antigen does not associate with the PDZ domain proteins ZO-1, AF-6, or ASIP/PAR-3, despite the presence of a PDZ-binding motif. The 1G8 antigen was not detected on peripheral blood mouse leukocytes, whereas similar to JAM-1 it was strongly expressed on platelets and megakaryocytes. The 1G8 antigen supports homophilic interactions on transfected Chinese hamster ovary cells. Based on the similarity to the JAM molecules, it is plausible that the 1G8 antigen might be involved in interendothelial cell adhesion.

Microfilament-dependent movement of the beta3 integrin subunit within focal contacts of endothelial cells.

To gain insight into the dynamic properties of focal contacts, we induced expression of green fluorescent protein-tagged beta3 integrin (GFP-beta3) and actinin-1 (GFP-actinin-1) in endothelial cells. Both tagged proteins localize with alpha(v)beta3 integrin in focal contacts distributed towards the periphery of transfected cells. Labeled focal contacts migrate at about 0.1 mm/min in stationary live endothelial cells. We compared beta3 integrin and actinin-1 dynamics in focal contacts by using fluorescence recovery after photobleaching. Recovery of signal in bleached focal contacts that have incorporated actinin-1 is rapid and occurs within less than 4 min. This recovery is energy-dependent. In contrast, recovery of bleached focal contacts that contain GFP-beta3 integrin takes longer than 30 min. Yet, when a narrow stripe of fluorescence is bleached across a beta3 integrin-labeled focal contact, recovery is complete within 16 min. The latter recovery is energy-dependent and is blocked not only by actin-filament disrupting drugs but also by a myosin light chain kinase inhibitor. Thus, integrins are not immobile when incorporated into focal contacts, as some have suggested. We propose that integrins are mobile within the confines of focal contacts and that this mobility is supported by an actin-associated molecular motor.

Dynamics of integrin clustering at focal contacts of endothelial cells studied by multimode imaging microscopy.

Human umbilical vein endothelial cells were stained with FITC-labeled anti-beta(1) integrin antibody and plated on a glass cover slip to elucidate the mechanism of integrin clustering during focal contact formation. The process of integrin clustering was observed by time-lapse total-internal-reflection fluorescence microscopy, which can selectively visualize the labeled integrins at the basal surface of living cells. The clustering of integrins at focal contacts started at 1 hour after plating and individual clusters kept growing for approximately 6 hours. Most integrin clusters (approximately 80%) elongated towards the cell center or along the cell margin at a rate of 0.29+/-0.24 microm minute(-1). Photobleaching and recovery experiments with evanescent illumination revealed that the integrins at the extending tip of the clusters were supplied from the intracellular space. Simultaneous time-lapse imaging of exocytosis of integrin-containing vesicles and elongating focal contacts showed that most exocytosis occurred at or near the focal contacts followed by their elongation. Double staining of F-actins and integrins demonstrated that stress fibers were located near the integrin clusters and that intracellular punctate integrins were associated with these stress fibers. These results suggest that the clustering of integrins is mediated by actin-fiber-dependent translocation of integrins to the extending tip of focal contacts.

Molecular mechanisms that control endothelial cell contacts.

Endothelial cell contacts control the permeability of the blood vessel wall. This allows the endothelium to form a barrier for solutes, macromolecules, and leukocytes between the vessel lumen and the interstitial space. Loss of this barrier function in pathophysiological situations can lead to extracellular oedema. The ability of leukocytes to enter tissue at sites of inflammation is dependent on molecular mechanisms that allow leukocytes to adhere to the endothelium and to migrate through the endothelial cell layer and the underlying basal lamina. It is a commonly accepted working hypothesis that inter-endothelial cell contacts are actively opened and closed during this process. Angiogenesis is another important process that requires well-controlled regulation of inter-endothelial cell contacts. The formation of new blood vessels by sprouting from pre-existing vessels depends on the loosening of established endothelial cell contacts and the migration of endothelial cells that form the outgrowing sprouts. This review focuses on the molecular composition of endothelial cell surface proteins and proteins of the cytoskeletal undercoat of the plasma membrane at sites of inter-endothelial cell contacts and discusses the current knowledge about the potential role of such molecules in the regulation of endothelial cell contacts.

Molecular mechanisms that control endothelial cell contacts

Endothelial cell contacts control the permeability of the blood vessel wall. This allows the endothelium to form a barrier for solutes, macromolecules, and leukocytes between the vessel lumen and the interstitial space. Loss of this barrier function in pathophysiological situations can lead to extracellular oedema. The ability of leukocytes to enter tissue at sites of inflammation is dependent on molecular mechanisms that allow leukocytes to adhere to the endothelium and to migrate through the endothelial cell layer and the underlying basal lamina. It is a commonly accepted working hypothesis that inter-endothelial cell contacts are actively opened and closed during this process. Angiogenesis is another important process that requires well-controlled regulation of inter-endothelial cell contacts. The formation of new blood vessels by sprouting from pre-existing vessels depends on the loosening of established endothelial cell contacts and the migration of endothelial cells that form the outgrowing sprouts. This review focuses on the molecular composition of endothelial cell surface proteins and proteins of the cytoskeletal undercoat of the plasma membrane at sites of inter-endothelial cell contacts and discusses the current knowledge about the potential role of such molecules in the regulation of endothelial cell contacts. Copyright © 2000 John Wiley & Sons, Ltd.

Vascular pathology: a pathogenetic challenge for a new millennium

Vascular pathology: a pathogenetic challenge for a new millennium