Ferruccio Breviario

Contact inhibition of VEGF-induced proliferation requires vascular endothelial cadherin, β-catenin, and the phosphatase DEP-1/CD148

Confluent endothelial cells respond poorly to the proliferative signals of VEGF. Comparing isogenic endothelial cells differing for vascular endothelial cadherin (VE-cadherin) expression only, we found that the presence of this protein attenuates VEGF-induced VEGF receptor (VEGFR) 2 phosphorylation in tyrosine, p44/p42 MAP kinase phosphorylation, and cell proliferation. VE-cadherin truncated in β-catenin but not p120 binding domain is unable to associate with VEGFR-2 and to induce its inactivation. β-Catenin–null endothelial cells are not contact inhibited by VE-cadherin and are still responsive to VEGF, indicating that this protein is required to restrain growth factor signaling. A dominant-negative mutant of high cell density–enhanced PTP 1 (DEP-1)//CD148 as well as reduction of its expression by RNA interference partially restore VEGFR-2 phosphorylation and MAP kinase activation. Overall the data indicate that VE-cadherin–β-catenin complex participates in contact inhibition of VEGF signaling. Upon stimulation with VEGF, VEGFR-2 associates with the complex and concentrates at cell–cell contacts, where it may be inactivated by junctional phosphatases such as DEP-1. In sparse cells or in VE-cadherin–null cells, this phenomenon cannot occur and the receptor is fully activated by the growth factor.

VEGF receptor 2 and the adherens junction as a mechanical transducer in vascular endothelial cells

Blood-flow interactions with the vascular endothelium represents a specialized example of mechanical regulation of cell function that has important physiological and pathophysiological cardiovascular consequences. Yet, the mechanisms of mechanostransduction are not understood fully. This study shows that shear stress induces a rapid induction as well as nuclear translocation of the vascular endothelial growth factor (VEGF) receptor 2 and promotes the binding of the VEGF receptor 2 and the adherens junction molecules, VE-cadherin and β-catenin, to the endothelial cytoskeleton. These changes are accompanied by the formation of a complex containing the VEGF receptor 2–VE-cadherin–β-catenin. In endothelial cells lacking VE-cadherin, shear stress did not augment nuclear translocation of the VEGF receptor 2 and phosphorylation of Akt1 and P38 as well as transcriptional induction of a reporter gene regulated by a shear stress-responsive promoter. These results suggest that VEGF receptor 2 and the adherens junction act as shear-stress cotransducers, mediating the transduction of shear-stress signals into vascular endothelial cells.

INHIBITION OF CULTURED CELL GROWTH BY VASCULAR ENDOTHELIAL CADHERIN (CADHERIN-5/VE-CADHERIN)

Endothelial cell proliferation is inhibited by the establishment of cell to cell contacts. Adhesive molecules at junctions could therefore play a role in transferring negative growth signals. The transmembrane protein VE-cadherin (vascular endothelial cadherin/cadherin-S) is selectively expressed at intercellular clefts in the endothelium. The intracellular domain interacts with cytoplasmic proteins called catenins that transmit the adhesion signal and contribute to the anchorage of the protein to the actin cytoskeleton. Transfection of VE-cadherin in both Chinese hamster ovary (CHO) and L929 cells confers inhibition of cell growth. Truncation of VE-cadherin cytoplasmic region, responsible for linking catenins, does not affect VE-cadherin adhesive properties but abolishes its effect on cell growth. Seeding human umbilical vein endothelial cells or VE-cadherin transfectants on a recombinant VE-cadherin amino-terminal fragment inhibited their proliferation. These data show that VE-cadherin homotypic engagement at junctions participates in density dependent inhibition of cell growth. This effect requires both the extracellular adhesive domain and the intracellular catenin binding region of the molecule.

Characterization of the Promoter for the Human Long Pentraxin PTX3 ROLE OF NF-κB IN TUMOR NECROSIS FACTOR-α AND INTERLEUKIN-1β REGULATION

The “long pentraxins” are an emerging family of genes that have conserved in their carboxy-terminal halves a pentraxin domain homologous to the prototypical acute phase protein pentraxins (C-reactive protein and serum amyloid P component) and acquired novel amino-terminal domains. In this report, a genomic fragment of 1371 nucleotides from the human “long pentraxin” gene PTX3 is characterized as a promoter on tumor necrosis factor-α (TNFα) and interleukin (IL)-1β exposure in transfected 8387 human fibroblasts by chloramphenicol acetyltransferase and RNase protection assays. In the same cells, the PTX3 promoter does not respond to IL-6 stimulation. Furthermore, IL-1β and TNFα responsiveness is not seen in the Hep 3B hepatoma cell line. The minimal promoter contains one NF-κB element which is shown to be necessary for induction and able to bind p50 homodimers and p65 heterodimers but not c-Rel. Mutants in this site lose the ability to bind NF-κB proteins and to respond to TNFα and IL-1β in functional assays. Sp1- and AP-1 binding sites lying in proximity to the NF-κB site do not seem to play a major role for cytokine responsiveness. Finally, cotransfection experiments with expression vectors validate that the natural promoter contains a functional NF-κB site.

Association of Junctional Adhesion Molecule with Calcium/calmodulin-dependent Serine Protein Kinase (CASK/LIN-2) in Human Epithelial Caco-2 Cells

We report here that junctional adhesion molecule (JAM) interacts with calcium/calmodulin-dependent serine protein kinase (CASK), a protein related to membrane-associated guanylate kinases. In Caco-2 cells, JAM and CASK were coprecipitated and found to colocalize at intercellular contacts along the lateral surface of the plasma membrane. Association of JAM with CASK requires the PSD95/dlg/ZO-1 (PDZ) domain of CASK and the putative PDZ-binding motif Phe-Leu-ValCOOH in the cytoplasmic tail of JAM. Temporal dissociation in the junctional localization of the two proteins suggests that the association with CASK is not required for recruiting JAM to intercellular junctions. Compared with mature intercellular contacts, junction assembly was characterized by both enhanced solubility of CASK in Triton X-100 and reduced amounts of Triton-insoluble JAM-CASK complexes. We propose that JAM association with CASK is modulated during junction assembly, when CASK is partially released from its cytoskeletal associations.

Sox17 is indispensable for acquisition and maintenance of arterial identity

The functional diversity of the arterial and venous endothelia is regulated through a complex system of signalling pathways and downstream transcription factors. Here we report that the transcription factor Sox17, which is known as a regulator of endoderm and hemopoietic differentiation, is selectively expressed in arteries, and not in veins, in the mouse embryo and in mouse postnatal retina and adult. Endothelial cell-specific inactivation of Sox17 in the mouse embryo is accompanied by a lack of arterial differentiation and vascular remodelling that results in embryo death in utero. In mouse postnatal retina, abrogation of Sox17 expression in endothelial cells leads to strong vascular hypersprouting, loss of arterial identity and large arteriovenous malformations. Mechanistically, Sox17 acts upstream of the Notch system and downstream of the canonical Wnt system. These data introduce Sox17 as a component of the complex signalling network that orchestrates arterial/venous specification.

Vascular Endothelial Growth Factor Induces Shc Association With Vascular Endothelial Cadherin. A Potential Feedback Mechanism to Control Vascular Endothelial Growth Factor Receptor-2 Signaling

Vascular endothelial (VE)-cadherin is endothelium specific, mediates homophilic adhesion, and is clustered at intercellular junctions. VE-cadherin is required for normal development of the vasculature in the embryo and for angiogenesis in the adult. Here, we report that VE-cadherin is associated with VE growth factor (VEGF) receptor-2 (VEGFR-2) on the exposure of endothelial cells to VEGF. The binding parallels receptor phosphorylation on tyrosine residues, which is maximal at 5 minutes and then declines within 30 minutes. Tyrosine phosphorylation of VE-cadherin was maximal at 30 minutes after the addition of the growth factor. At this time point, the protein could be coimmunoprecipitated with the adaptor protein Shc. Pull-down experiments with different Shc domains and mutants of the VE-cadherin cytoplasmic tail have shown that Shc binds to the carboxy-terminal domain of the VE-cadherin tail through its Src homology 2 domain (SH2). We found that Shc phosphorylation lasts longer in endothelial cells carrying a targeted null mutation in the VE-cadherin gene than in VE-cadherin–positive cells. These data suggest that VE-cadherin expression exerts a negative effect on Shc phosphorylation by VEGFR-2. We speculate that VE-cadherin binding to Shc promotes its dephosphorylation through associated phosphatases.

Identification of a Novel Cadherin (Vascular Endothelial Cadherin-2) Located at Intercellular Junctions in Endothelial Cells

Endothelial cells express two major cadherins, VE- and N-cadherins, but only the former consistently participates in adherens junction organization. In heart microvascular endothelial cells, we identified a new member of the cadherin superfamily using polymerase chain reaction. The entire putative coding sequence was determined. Similarly to protocadherins, while the extracellular domain presented homology with other members of the cadherin superfamily, the intracellular region was unrelated either to cadherins or to any other known protein. We propose for this new protein the name of vascular endothelial cadherin-2. By Northern blot analysis, the mRNA was present only in cultured endothelial cell lines but not in other cell types such as NIH 3T3, Chinese hamster ovary, or L cells. In addition, mRNA was particularly abundant in highly vascularized organs such as lung or kidney. In endothelial cells and transfectants, this cadherin was unable to bind catenins and presented a weak association with the cytoskeleton. This new molecule shares some functional properties with VE-cadherin and other members of the cadherin family. In Chinese hamster ovary transfectants it promoted homotypic Ca2+ dependent aggregation and adhesion and clustered at intercellular junctions. However, in contrast to VE-cadherin, it did not modify paracellular permeability, cell migration, and density-dependent cell growth. These observations suggest that different cadherins may promote homophilic cell-to-cell adhesion but that the functional consequences of this interaction depend on their binding to specific intracellular signaling/cytoskeletal proteins.

1 Endothelial cell-to-cell junctions. Structural characteristics and functional role in the regulation of vascular permeability and leukocyte extravasation*

Endothelial monolayer forms the main barrier to the passage of macromolecules and circulating cells from blood to tissues. This property is regulated by intercellular junctions. These are complex structures formed by transmembrane adhesive molecules linked to a network of cytoplasmic cytoskeletal proteins. Endothelial junctions vary in number and organization along the vascular tree. Some transmembrane components of endothelial junctions have recently been identified. One is specifically expressed by endothelial cells (cadherin-5) while others (such as PECAM-1 and integrins) are also present in other cell types. The mechanisms that regulate the opening and the closure of endothelial junctions are still obscure. It is possible that inflammatory agents increase permeability by binding to specific receptors on the endothelial membrane. This would lead to the generation of intracellular signals causing cytoskeletal reorganization and opening of interendothelial gaps. This general sequence of events, however, seems to follow specific routes for different stimuli. In fact, permeability-increasing agents differ in the type of intracellular second messenger they trigger, for the time course of their effect, and for their specificity for the endothelium of different vascular districts. Endothelial junctions also regulate leukocyte extravasation. Endothelial cells actively contribute to this process by expressing adhesive molecules on their surface and by releasing chemotactic cytokines. Once leukocytes have adhered to the endothelium, a coordinated opening of interendothelial junctions occurs. The mechanism by which this takes place is unknown but it might resemble that triggered by soluble inflammatory mediators.

Rapid retroviral infection of human haemopoietic cells of different lineages: efficient transfer in fresh T cells

In order to develop a clinically feasible gene marking approach, we have used the recently described PINCO retroviral expression system, composed of the enhanced green fluorescence protein (EGFP) cDNA driven by Moloney MLV LTR and packaged in the Phoenix amphotropic cell line. Two T, five B, one erythromyeloid and three myeloid cell lines were successfully infected with % GFP+ cells ranging from 4% to 79%, showing a lineage‐dependent difference in infection susceptibility, with the myeloid cells being the least efficiently infected. We also infected normal mononuclear peripheral cells cultured in PHA and rhIL‐2 for 2 d, and obtained an average of 30% GFP+ cells, all present within the CD3+ population, with CD4+ and CD8+ cells being equally infected. Finally, the tonsillar purified B population showed lower levels of infectivity (6%) whereas high susceptibility was shown by normal human umbilical vein endothelial cells (57%). Highly purified CD34+ cells were also susceptible, varying from 6% to 10% GFP+ cells. Immature myeloid/erythroid progenitors have been infected which stably expressed the GFP protein during further differentiation in culture. The GFP+ T cells were FACS‐sorted rapidly upon infection, subsequently cultured and the fluorescence intensity monitored. In all cases the difference in percentage of GFP+ cells did not correlate with the percentage of S/G2/M cycling cells as determined at the moment of infection or with the expression levels of Ram‐1 amphotrophic receptor. The improved safety of this retroviral system, the rapidity of the technique, the high efficiency of infection with respect to normal T lymphocytes (in this last case higher than previously reported) and the lack of need for in vitro selection make this system favourable for clinical development.