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Elisabetta Dejana

Publications and source records attributed to Elisabetta Dejana.

At least 19 recordsLinked to original sources
PubMed2006

Vascular endothelial cadherin controls VEGFR-2 internalization and signaling from intracellular compartments.

Receptor endocytosis is a fundamental step in controlling the magnitude, duration, and nature of cell signaling events. Confluent endothelial cells are contact inhibited in their growth and respond poorly to the proliferative signals of vascular endothelial growth factor (VEGF). In a previous study, we found that the association of vascular endothelial cadherin (VEC) with VEGF receptor (VEGFR) type 2 contributes to density-dependent growth inhibition (Lampugnani, G.M., A. Zanetti, M. Corada, T. Takahashi, G. Balconi, F. Breviario, F. Orsenigo, A. Cattelino, R. Kemler, T.O. Daniel, and E. Dejana. 2003. J. Cell Biol. 161:793-804). In the present study, we describe the mechanism through which VEC reduces VEGFR-2 signaling. We found that VEGF induces the clathrin-dependent internalization of VEGFR-2. When VEC is absent or not engaged at junctions, VEGFR-2 is internalized more rapidly and remains in endosomal compartments for a longer time. Internalization does not terminate its signaling; instead, the internalized receptor is phosphorylated, codistributes with active phospholipase C-gamma, and activates p44/42 mitogen-activated protein kinase phosphorylation and cell proliferation. Inhibition of VEGFR-2 internalization reestablishes the contact inhibition of cell growth, whereas silencing the junction-associated density-enhanced phosphatase-1/CD148 phosphatase restores VEGFR-2 internalization and signaling. Thus, VEC limits cell proliferation by retaining VEGFR-2 at the membrane and preventing its internalization into signaling compartments.

Antigens, CD↗
PubMed2006

The role of JAM-A and PECAM-1 in modulating leukocyte infiltration in inflamed and ischemic tissues.

Innate and adaptive immunological responses are accompanied by leukocyte adhesion to the blood-vessel wall and their subsequent infiltration into the underlying tissues. In the majority of the cases, leukocytes cross the endothelium by squeezing through the border of apposed endothelial cells, a process that is known as diapedesis. Many data suggest that proteins at endothelial junctions establish homophilic interactions with identical proteins, which are present on leukocytes. These interactions might then direct the passage of leukocytes through the endothelial border. In this review, we focus on two endothelial junctional proteins [junctional adhesion molecule-A (JAM-A) and PECAM], which play an important role in leukocyte diapedesis. In vivo data with blocking antibodies or inactivation of JAM-A and PECAM genes indicate that the role of these two proteins depends on the stimulus and the experimental model used.

Animals↗
PubMed2006

Increase in vascular permeability and vasodilation are critical for proangiogenic effects of stem cell therapy.

BACKGROUND: Proangiogenic cell therapy based on administration of bone marrow-derived mononuclear cells (BMCs) or endothelial progenitor cells (EPCs) is now under investigation in humans for the treatment of ischemic diseases. However, mechanisms leading to the beneficial effects of BMCs and EPCs remain unclear. METHODS AND RESULTS: BMC- and CD34+-derived progenitor cells interacted with ischemic femoral arteries through SDF-1 and CXCR4 signaling and released nitric oxide (NO) via an endothelial nitric oxide synthase (eNOS)-dependent pathway. BMC-induced NO production promoted a marked vasodilation and disrupted vascular endothelial-cadherin/beta-catenin complexes, leading to increased vascular permeability. NO-dependent vasodilation and hyperpermeability were critical for BMC infiltration in ischemic tissues and their proangiogenic potential in a model of hindlimb ischemia in mice. CONCLUSIONS: Our results propose a new concept that proangiogenic progenitor cell activity does not rely only on their ability to differentiate into endothelial cells but rather on their capacity to modulate the function of preexisting vessels.

Animals↗
PubMed2006

The multiple languages of endothelial cell-to-cell communication.

Intercellular adhesion plays a key role during development and maintenance of tissue homeostasis. Within the vascular system, cell-cell adhesion is particularly important for the correct formation, networking, and remodeling of vessels. Although in vascular endothelial cells adhesive junctions account for the integrity of the vessel wall, they are not to be considered as static molecular structures that function as intercellular glue. This becomes evident during the remodeling of the endothelium in various physiological and pathological processes, requiring highly dynamic vascular adhesion complexes. Moreover, it has recently become evident that, besides their structural functions, adhesion molecules involved in endothelial cell-cell interaction play an important role in inducing and integrating intracellular signals that, in turn, impact on several aspects of vascular cell physiology. In this review, we describe these recent findings focusing on junctional proteins at adherens and tight junctions. The role of this adhesion molecule-mediated signaling is discussed in the context of developmental and pathological angiogenesis.

Adherens Junctions↗
PubMed2006

Generation and characterization of a mouse lymphatic endothelial cell line.

Lymphatic vessels, by channeling fluid and leukocytes from the periphery into lymph nodes, play a central role in the development of the immune response. Despite their importance in homeostasis and disease, the difficulties in enriching and culturing lymphatic endothelial cells limit studies of their biology. Here, we report the isolation, stabilization, and characterization of a mouse lymphatic endothelial cell line (MELC) and the generated clones thereof. Cells were isolated from benign lymphangiomas induced by intraperitoneal injections of incomplete Freund's adjuvant. The MELC line expressed molecules typical of lymphatic endothelium, including VEGFR3/Flt-4, podoplanin, Prox-1, and D6, but not LYVE-1. It also expressed CD34, ICAM-1, VCAM, and JAM-A, but not CD31, VE-cadherin, E-selectin, or CX3CL1/fractalkine (both TNFalpha-induced), at variance with vascular endothelial cells tested in parallel. The inflammatory cytokines TNFalpha and IL-4 regulated production of selected adhesion molecules (VCAM), cytokines (IL-6), and chemokines (CCL2/JE). Whole genome transcriptional profiling identified a set of 150 known genes differentially expressed in MELC versus vascular endothelial cells. Thus, the MELC line may represent an invaluable source of lymphatic endothelium.

Animals↗
PubMed2005

Importance of junctional adhesion molecule-A for neointimal lesion formation and infiltration in atherosclerosis-prone mice.

OBJECTIVE: Although junctional adhesion molecule-A (JAM-A) has recently been implicated in leukocyte recruitment on early atherosclerotic endothelium and after reperfusion injury, its role in neointima formation after arterial injury remains to be elucidated. METHODS AND RESULTS: Here we show that the genetic deletion of JAM-A in apolipoprotein E-deficient (apoE(-/-)) mice significantly reduced neointimal hyperplasia after wire injury of carotid arteries without altering medial area. This was associated with a significant decrease in neointimal macrophage content, whereas the relative content of smooth muscle cells and endothelial recovery was unaltered in JAM-A(-/-)apoE(-/-) compared with JAM-A(+/+)apoE(-/-) lesions. In carotid arteries perfused ex vivo, deficiency in JAM-A significantly impaired the recruitment of monocytes 1 week, but not 1 day, after injury. These effects were paralleled by an attenuation of monocyte arrest and transmigration on activated JAM-A(-/-)apoE(-/-) versus JAM-A(+/+)apoE(-/-) endothelial cells under flow conditions in vitro. A mechanism underlying reduced recruitment was implied by findings that the luminal expression of the arrest chemokine RANTES in injured arteries and its endothelial deposition by activated platelets in vitro were diminished by JAM-A deficiency. CONCLUSIONS: Our data provide the first evidence to our knowledge for a crucial role of JAM-A in accelerated lesion formation and monocyte infiltration in atherosclerosis-prone mice.

Animals↗
PubMed2005

Endothelial cadherins and tumor angiogenesis.

Adherens junctions and more specifically cadherins play an important role in endothelial cell integrity and growth and, in general, in vascular morphogenesis. Besides their adhesive properties, cadherins may act by transferring intracellular signals through interaction with a complex network of cytoskeletal and signaling molecules. Cadherins may signal in different ways: through direct activation of signaling pathways, through interaction with cell-specific growth factor receptors or by controlling beta-catenin and/or other transcription factors' translocation to the nucleus. Endothelial cells present different cadherins which may transfer specific signals and exert distinct functional roles. VE-cadherin is endothelial-specific and the major constituent of adherens junctions. This protein is able to protect endothelial cells from apoptosis and contributes to contact inhibition of endothelial cell growth. N-cadherin is also abundantly expressed in the endothelium and may be important in modulating VE-cadherin expression. T cadherin, R-cadherin and VE-cadherin 2 were found in specific regions of the vascular tree but their role in vascular development or angiogenesis is still unclear.

Animals↗
PubMed2005

Epac1 regulates integrity of endothelial cell junctions through VE-cadherin.

We have previously shown that Rap1 as well as its guanine nucleotide exchange factor Epac1 increases cell-cell junction formation. Here, we show that activation of Epac1 with the exchange protein directly activated by cAMP (Epac)-specific cAMP analog 8CPT-2'O-Me-cAMP (007) resulted in a tightening of the junctions and a decrease in the permeability of the endothelial cell monolayer. In addition, 007 treatment resulted in the breakdown of actin stress fibers and the formation of cortical actin. These effects were completely inhibited by siRNA against Epac1. In VE-cadherin knock-out cells Epac1 did not affect cell permeability, whereas in cells re-expressing VE-cadherin this effect was restored. Finally, the effect of Epac activation on the actin cytoskeleton was independent of junction formation. From these results we conclude that in human umbilical vein endothelial cells Epac1 controls VE-cadherin-mediated cell junction formation and induces reorganization of the actin cytoskeleton.

Actins↗
PubMed2005

p120-Catenin regulates clathrin-dependent endocytosis of VE-cadherin.

VE-cadherin is an adhesion molecule critical to vascular barrier function and angiogenesis. VE-cadherin expression levels are regulated by p120 catenin, which prevents lysosomal degradation of cadherins by unknown mechanisms. To test whether the VE-cadherin cytoplasmic domain mediates endocytosis, and to elucidate the nature of the endocytic machinery involved, the VE-cadherin tail was fused to the interleukin (IL)-2 receptor (IL-2R) extracellular domain. Internalization assays demonstrated that the VE-cadherin tail dramatically increased endocytosis of the IL-2R in a clathrin-dependent manner. Interestingly, p120 inhibited VE-cadherin endocytosis via a mechanism that required direct interactions between p120 and the VE-cadherin cytoplasmic tail. However, p120 did not inhibit transferrin internalization, demonstrating that p120 selectively regulates cadherin internalization rather than globally inhibiting clathrin-dependent endocytosis. Finally, cell surface labeling experiments in cells expressing green fluorescent protein-tagged p120 indicated that the VE-cadherin-p120 complex dissociates upon internalization. These results support a model in which the VE-cadherin tail mediates interactions with clathrin-dependent endocytic machinery, and this endocytic processing is inhibited by p120 binding to the cadherin tail. These findings suggest a novel mechanism by which a cytoplasmic binding partner for a transmembrane receptor can serve as a selective plasma membrane retention signal, thereby modulating the availability of the protein for endo-lysosomal processing.

Adolescent↗
PubMed2005

Junctional adhesion molecule-A-deficient polymorphonuclear cells show reduced diapedesis in peritonitis and heart ischemia-reperfusion injury.

Junctional Adhesion Molecule-A (JAM-A) is a transmembrane adhesive protein expressed at endothelial junctions and in leukocytes. Here we report that JAM-A is required for the correct infiltration of polymorphonuclear leukocytes (PMN) into an inflamed peritoneum or in the heart upon ischemia-reperfusion injury. The defect was not observed in mice with an endothelium-restricted deficiency of the protein but was still detectable in mice transplanted with bone marrow from JAM-A(-/-) donors. Microscopic examination of mesenteric and heart microvasculature of JAM-A(-/-) mice showed high numbers of PMN adherent on the endothelium or entrapped between endothelial cells and the basement membrane. In vitro, in the absence of JAM-A, PMN adhered more efficiently to endothelial cells and basement membrane proteins, and their polarized movement was strongly reduced. This paper describes a nonredundant role of JAM-A in controlling PMN diapedesis through the vessel wall.

Animals↗
PubMed2005

Histone deacetylase activity is essential for the expression of HoxA9 and for endothelial commitment of progenitor cells.

The regulation of acetylation is central for the epigenetic control of lineage-specific gene expression and determines cell fate decisions. We provide evidence that the inhibition of histone deacetylases (HDACs) blocks the endothelial differentiation of adult progenitor cells. To define the mechanisms by which HDAC inhibition prevents endothelial differentiation, we determined the expression of homeobox transcription factors and demonstrated that HoxA9 expression is down-regulated by HDAC inhibitors. The causal involvement of HoxA9 in the endothelial differentiation of adult progenitor cells is supported by the finding that HoxA9 overexpression partially rescued the endothelial differentiation blockade induced by HDAC inhibitors. Knockdown and overexpression studies revealed that HoxA9 acts as a master switch to regulate the expression of prototypical endothelial-committed genes such as endothelial nitric oxide synthase, VEGF-R2, and VE-cadherin, and mediates the shear stress-induced maturation of endothelial cells. Consistently, HoxA9-deficient mice exhibited lower numbers of endothelial progenitor cells and showed an impaired postnatal neovascularization capacity after the induction of ischemia. Thus, HoxA9 is regulated by HDACs and is critical for postnatal neovascularization.

Animals↗
PubMed2005

Endoglin null endothelial cells proliferate faster and are more responsive to transforming growth factor beta1 with higher affinity receptors and an activated Alk1 pathway.

Endoglin is an accessory receptor for transforming growth factor beta (TGFbeta) in endothelial cells, essential for vascular development. Its pivotal role in angiogenesis is underscored in Endoglin null (Eng-/-) murine embryos, which die at mid-gestation (E10.5) from impaired yolk sac vessel formation. Moreover, mutations in endoglin and the endothelial-specific TGFbeta type I receptor, ALK1, are linked to hereditary hemorrhagic telangiectasia. To determine the role of endoglin in TGFbeta pathways, we derived murine endothelial cell lines from Eng+/+ and Eng-/- embryos (E9.0). Whereas Eng+/+ cells were only partially growth inhibited by TGFbeta, Eng-/- cells displayed a potent anti-proliferative response. TGFbeta-dependent Smad2 phosphorylation and Smad2/3 translocation were unchanged in the Eng-/- cells. In contrast, TGFbeta treatment led to a more rapid activation of the Smad1/5 pathway in Eng null cells that was apparent at lower TGFbeta concentrations. Enhanced activity of the Smad1 pathway in Eng-/- cells was reflected in higher expression of ALK1-dependent genes such as Id1, Smad6, and Smad7. Analysis of cell surface receptors revealed that the TGFbeta type I receptor, ALK5, which is required for ALK1 function, was increased in Eng-/- cells. TGFbeta receptor complexes were less numerous but displayed a higher binding affinity. These results suggest that endoglin modulates TGFbeta signaling in endothelial cells by regulating surface TGFbeta receptors and suppressing Smad1 activation. Thus an altered balance in TGFbeta receptors and downstream Smad pathways may underlie defects in vascular development and homeostasis.

Active Transport, Cell Nucleus↗
PubMed2005

Junctional adhesion molecule-A deficiency increases hepatic ischemia-reperfusion injury despite reduction of neutrophil transendothelial migration.

The endothelial receptors that control leukocyte transmigration in the postischemic liver are not identified. We investigated the role of junctional adhesion molecule-A (JAM-A), a receptor expressed in endothelial tight junctions, leukocytes, and platelets, for leukocyte transmigration during hepatic ischemia-reperfusion (I/R) in vivo. We show that JAM-A is up-regulated in hepatic venular endothelium during reperfusion. I/R-induced neutrophil transmigration was attenuated in both JAM-A-/- and endothelial JAM-A-/- mice as well as in mice treated with an anti-JAM-A antibody, whereas transmigration of T cells was JAM-A independent. Postischemic leukocyte rolling remained unaffected in JAM-A-/- and endothelial JAM-A-/- mice, whereas intravascular leukocyte adherence was increased. The extent of interactions of JAM-A-/- platelets with the postischemic endothelium was comparable with that of JAM-A+/+ platelets. The I/R-induced increase in the activity of alanine aminotransferase (ALT)/aspartate aminotransferase (AST) and sinusoidal perfusion failure was not reduced in JAM-A-/- mice, while the number of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL)-positive hepatocytes was significantly higher. Thus, we show for the first time that JAM-A is up-regulated in hepatic venules and serves as an endothelial receptor of neutrophil transmigration, but it does not mediate leukocyte rolling, adhesion, or platelet-endothelial cell interactions. JAM-A deficiency does not reduce I/R-induced microvascular and hepatocellular necrotic injury, but increases hepatocyte apoptosis, despite attenuation of neutrophil infiltration.

Animals↗
PubMed2005

Mesoangioblasts, vessel-associated multipotent stem cells, repair the infarcted heart by multiple cellular mechanisms: a comparison with bone marrow progenitors, fibroblasts, and endothelial cells.

OBJECTIVE: To test the potential of mesoangioblasts (Mabs) in reducing postischemic injury in comparison with bone marrow progenitor cells (BMPCs), fibroblasts (Fbs), and embryonic stem cell-derived endothelial cells (ECs), and to identify putative cellular protective mechanisms. METHODS AND RESULTS: Cells were injected percutaneously in the left ventricular (LV) chamber of C57BL/6 mice, 3 to 6 hours after coronary ligation, and detected in the hearts 2 days and 6 weeks later. Echocardiographic examinations were performed at 6 weeks. LV dilation was reduced and LV shortening fraction was improved with Mabs and BMPCs but not with ECs and Fbs. Donor cell colonization of the host myocardium was modest and predominantly in the smooth muscle layer of vessels. Capillary density was higher in the peripheral infarct area and apoptotic cardiomyocytes were fewer with Mabs and BMPCs. Mabs and BMPCs, but not Fbs or ECs, promoted survival of cultured cardiocytes under low-oxygen in culture. This activity was present in Mab-conditioned medium and could be replaced by a combination of basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF)-1, and hepatocyte growth factor (HGF), all of which are produced by these cells. Conditioned medium from Mabs, but not from Fbs, stimulated proliferation of smooth muscle cells in vitro. CONCLUSIONS: Mabs appear as effective as BMPCs in reducing postinfarction LV dysfunction, likely through production of antiapoptotic and angiogenic factors.

Animals↗
PubMed2005

Expression of junctional adhesion molecule-A prevents spontaneous and random motility.

Junctional adhesion molecule-A (JAM-A) is a cell-surface glycoprotein that localizes to intercellular junctions and associates with intracellular proteins via PSD95-Dlg-ZO1-binding residues. To define the functional consequences of JAM-A expression, we have produced endothelial cells from JAM-A-deficient mice. We report here that the absence of JAM-A enhanced spontaneous and random motility. In turn, the enhanced motility of JAM-A-negative cells was abrogated either on transfection of exogenous JAM-A or on treatment with inhibitors of glycogen synthase kinase-3beta (GSK-3beta). In addition, in JAM-A-positive cells, motility was enhanced on inactivation of protein kinase Czeta (PKCzeta), which is an inhibitor of GSK-3beta. Although these findings suggested that JAM-A might inhibit GSK-3beta, we found that expression per se of JAM-A did not change the levels of inactive GSK-3beta. Thus, JAM-A expression may regulate effectors of motility that are also downstream of the PKCzeta/GSK-3beta axis. In support of this view, we found that JAM-A absence increased the number of actin-containing protrusions, reduced the stability of microtubules and impaired the formation of focal adhesions. Notably, all the functional consequences of JAM-A absence were reversed either on treatment with GSK-3beta inhibitors or on transfection of full-length JAM-A, but not on transfection of a JAM-A deletion mutant devoid of the PSD95-Dlg-ZO1-binding residues. Thus, by regulating cytoskeletal and adhesive structures, JAM-A expression prevents cell motility, probably in a PSD95-Dlg-ZO1-dependent manner.

Animals↗
PubMed2005

Opposite effects of tumor necrosis factor and soluble fibronectin on junctional adhesion molecule-A in endothelial cells.

Junctional adhesion molecule-A (JAM-A) regulates key inflammatory responses, such as edema formation and leukocyte transmigration. Although it has been reported that the inflammatory cytokine tumor necrosis factor (TNF) causes the disassembly of JAM-A from the intercellular junctions, the mechanism has not been elucidated fully. Here, we report that TNF enhances the solubility of JAM-A in Triton X-100 and increases the amount of Triton-soluble JAM-A dimers at the cell surface but does not change the total levels of cellular JAM-A. Thus we hypothesized that TNF causes the redistribution of JAM-A from the junctions to the cell surface and that junction disassembly is sufficient to account for JAM-A redistribution. Intriguingly, however, even after complete disassembly of the junctions (with EDTA and trypsin), higher levels of JAM-A are detectable at the cell surface (by FACS analysis) in cells that had been previously incubated in the presence of TNF than in its absence. Thus we propose that TNF causes not only the disassembly of JAM-A from the junctions and its subsequent redistribution to the cell surface but also its dispersal in such a way that JAM-A becomes more easily accessible to the antibodies used for FACS analysis. Finally, we evaluated whether soluble fibronectin might attenuate the effects of TNF on JAM-A, as some inflammatory conditions are associated with the depletion of plasma fibronectin. We found that fibronectin reduces the effect of TNF on the disassembly of JAM-A, but not on its dispersal, thus further stressing that disassembly and dispersal can be functionally dissociated.

Animals↗