Distinct hemogenic potential of endothelial cells and CD41+ cells in mouse embryos

Definitive hematopoietic progenitor cells have been thought to develop from the vascular endothelium located in the aorta-gonad-mesonephros region of the mouse embryo. However, several recent findings have suggested that most hematopoietic progenitors are derived from non-endothelial precursor cells expressing CD41. We characterized two distinct precursor populations of definitive hematopoietic cell lineages, vascular endothelial (VE)-cadherin(+) CD41(-) CD45(-) endothelial cells and CD41(+) CD45(-) non-endothelial progenitors, both of which are derived from lateral mesoderm. VE-cadherin(+) endothelial cells obtained from cultures of differentiating embryonic stem cells possessed hematopoietic potential encompassing erythroid, myeloid and B lymphoid lineages, whereas CD41(+) progenitors lacked the B lymphopoietic potential. VE-cadherin(+) endothelial cells in the lower trunk of the embryo proper showed a significant potential for initiating B lymphopoiesis in cultures, while endothelial cells in the yolk sac appeared to have a bias for myeloerythropoietic differentiation. CD41(+) progenitors isolated from yolk sac and embryo proper were capable of generating multiple hematopoietic lineages, although mast cell precursors were exclusively enriched in CD41(+) progenitors in the yolk sac. These results suggest that hemogenic endothelial cells and CD41(+) progenitors possess distinct hematopoietic potential depending on the tissues in which they reside.     

Dynamics of vascular endothelial-cadherin and beta-catenin localization by vascular endothelial growth factor-induced angiogenesis in human umbilical vein cells

The adherens junctional molecule, vascular endothelial cadherin (VE-cadherin), functions to maintain adherens junction stability and to suppress apoptosis of endothelial cells by forming a complex with vascular endothelial growth factor (VEGF) receptor 2 and members of the armadillo family of cytoplasmic proteins. In order to investigate the dynamics of the association of VE-cadherin with adherens junctions during the initial stages of angiogenesis, human umbilical cord endothelial cells (HUVECs) were stimulated with VEGF to undergo angiogenesis in type-I collagen three-dimensional culture. In confluent monolayers of HUVECs, VE-cadherin and its signaling partner, beta-catenin, as well as the paracellular transmembrane adhesion molecule platelet-endothelial cell adhesion molecule (PECAM-1), were all present in regions of cell-cell contact. Within 3 h of stimulation of angiogenesis, VE-cadherin and beta-catenin were lost from these regions. In contrast, the distribution pattern of PECAM-1 did not alter. After 6 h the majority of endothelial cells had migrated to form a network of capillary cords with cell-cell contacts that contained all three molecules. By metabolic labeling of HUVECs it was found that de novo synthesis of VE-cadherin was not essential for the formation of new adherens junctions. Coimmunoprecipitation and immunoblotting experiments showed that the VE-cadherin and beta-catenin remained associated after they were lost from adherens junctions. Detergent extraction of cells with Triton X-100 indicted that the majority of VE-cadherin and beta-catenin was Triton soluble, indicating that they are only weakly associated with the actin-based cytoskeleton.     

Progressive divergence of definitive haematopoietic stem cells from the endothelial compartment does not depend on contact with the foetal liver

The yolk sac and the para-aortic splanchnopleura/aorta-genital ridges-mesonephros (P-Sp/AGM) region are the main sites of haematopoietic activity in the mouse embryo at the pre-liver stage of development. By day 11.5 of gestation, the AGM region is capable of autonomous initiation and expansion of definitive haematopoietic stem cells (HSCs). By day 12.5, HSC activity in the AGM region is reduced whilst a second wave of HSCs begins to emerge in the yolk sac. We show here that HSCs emerging in both locations are marked by co-expression of the endothelial-specific marker VE-cadherin and the pan-leukocyte antigen CD45. Phenotypic characterisation using CD31, TIE2, FLK1, Ac-LDL receptors, and CD34 markers demonstrated significant similarities between this VE-cadherin+CD45+ ;double-positive' population and endothelial cells suggesting a common origin for these cells. The double-positive fraction also expressed the stem cell markers Kit, Sca1 and AA4.1. Long-term transplantation experiments demonstrated that the double-positive population, which constituted less than 0.05% of the day 11.5 AGM region and the day 12.5 yolk sac, is highly enriched for HSCs. In vitro assays showed that this population is also enriched for myeloid progenitors. During foetal liver colonization, circulating HSCs remained within the VE-cadherin+ cell fraction, although their phenotypic similarity with endothelial cells became less prominent. Upon liver colonisation the majority of HSCs downregulated VE-cadherin, expression of which was completely lost in the adult bone marrow. Partial loss of VE-cadherin expression in HSCs can be observed extra hepatically in the advanced AGM region by E12.5. Similarly, the CD34+KIT+ population in the placenta, recently identified as a reservoir of HSCs, partly lose VE-cadherin expression by E12.5. By culturing isolated E11.5 AGM region and E12.5 yolk sac we show that the developmental switch from a ;primary' VE-cadherin+CD45+ to a more ;advanced' VE-cadherin-CD45+ phenotype does not require contact of HSCs with the liver and is probably a function of developmental time.     

Localization of claudin-5 and ZO-1 in rat spleen sinus endothelial cells

Splenic sinus endothelial cells, which adhere through tight and adherens junctions, regulate the passage of blood cells through the splenic cord. The objective of this study was to assess the localization of tight junctional proteins, claudin-5 and ZO-1 in the sinus endothelial cells of rat spleen and to characterize spatial and functional relationships between tight and adherens junctions. Immunofluorescence microscopy of tissue cryosections demonstrated that claudin-5, ZO-1, and α-catenin were distinctly localized in the junctional regions of adjacent endothelial cells. Immunogold electron microscopy demonstrated claudin-5 localized in the tight-junctional fused membranes of adjacent endothelial cells. Immunogold labeling for ZO-1 was localized not only in the tight-junctional-fused membranes of endothelial cells but also in the junctional membrane. α-Catenin was intermittently localized along the juxtaposed junctional membranes of adjacent endothelial cells. Double-staining immunogold microscopy for claudin-5 and ZO-1, claudin-5 and VE-cadherin, ZO-1 and VE-cadherin, and ZO-1 and α-catenin demonstrated that ZO-1 was closely localized to VE-cadherin and α-catenin in their juxtaposed membranes of endothelial cells. Thus, ZO-1 might play an important role in regulating the cell–cell junctions of sinus endothelial cells for blood–cell passage through splenic cords. This work was supported by a Grant-in-Aid for Scientific Research (C), Japan.     

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.     

VE-PTP and VE-cadherin ectodomains interact to facilitate regulation of phosphorylation and cell contacts

VE-cadherin is the essential adhesion molecule in endothelial adherens junctions, and the regulation of protein tyrosine phosphorylation is thought to be important for the control of adherens junction integrity. We show here that VE-PTP (vascular endothelial protein tyrosine phosphatase), an endothelial receptor-type phosphatase, co-precipitates with VE-cadherin, but not with beta-catenin, from cell lysates of transfected COS-7 cells and of endothelial cells. Co-precipitation of VE-cadherin and VE-PTP required the most membrane-proximal extracellular domains of each protein. Expression of VE-PTP in triple-transfected COS-7 cells and in CHO cells reversed the tyrosine phosphorylation of VE-cadherin elicited by vascular endothelial growth factor receptor 2 (VEGFR-2). Expression of VE-PTP under an inducible promotor in CHO cells transfected with VE-cadherin and VEGFR-2 increased the VE-cadherin-mediated barrier integrity of a cellular monolayer. Surprisingly, a catalytically inactive mutant form of VE-PTP had the same effect on VE-cadherin phosphorylation and cell layer permeability. Thus, VE-PTP is a transmembrane binding partner of VE-cadherin that associates through an extracellular domain and reduces the tyrosine phosphorylation of VE-cadherin and cell layer permeability independently of its enzymatic activity.     

A distinct PAR complex associates physically with VE-cadherin in vertebrate endothelial cells

A cell polarity complex consisting of partitioning defective 3 (PAR-3), atypical protein kinase C (aPKC) and PAR-6 has a central role in the development of cell polarity in epithelial cells. In vertebrate epithelial cells, this complex localizes to tight junctions. Here, we provide evidence for the existence of a distinct PAR protein complex in endothelial cells. Both PAR-3 and PAR-6 associate directly with the adherens junction protein vascular endothelial cadherin (VE-cadherin). This association is direct and mediated through non-overlapping domains in VE-cadherin. PAR-3 and PAR-6 are recruited independently to cell-cell contacts. Surprisingly, the VE-cadherin-associated PAR protein complex lacks aPKC. Ectopic expression of VE-cadherin in epithelial cells affects tight junction formation. Our findings suggest that in endothelial cells, another PAR protein complex exists that localizes to adherens junctions and does not promote cellular polarization through aPKC activity. They also point to a direct role of a cadherin in the regulation of cell polarity in vertebrates.     

Distribution of adherens junction mediated by VE-cadherin complex in rat spleen sinus endothelial cells

The splenic sinus endothelium regulates the passage of blood cells through the splenic cord. The goal of the present study was to assess the localization of vascular endothelial (VE)-cadherin, β-catenin, and p120-catenin in the sinus endothelial cells of rat spleen and to characterize the presence and distribution of adherens junction formation mediated by the cadherin-catenin complex. Immunofluorescent microscopy of tissue cryosections demonstrated that VE-cadherin, β-catenin, and p120-catenin were localized in the junctional regions of adjacent endothelial cells. Double-staining immunofluorescent microscopy for VE-cadherin and β-catenin revealed colocalization at junctional regions. Transmission electron microscopy of thin sections of sinus endothelial cells treated with Triton X-100 clearly showed adherens junctions within the plasma membrane. Adherens junctions were located at various levels in the lateral membranes of adjacent endothelial cells regardless of the presence or absence of underlying ring fibers. Immunogold electron microscopy revealed VE-cadherin, β-catenin, and p120-catenin in the juxtaposed junctional membranes of adjacent sinus endothelial cells. Double-staining immunogold microscopy for VE-cadherin and β-catenin and for VE-cadherin and p120-catenin demonstrated colocalization to the junctional membranes of adjacent endothelial cells. Immunolabeling was evident at various levels in the lateral junctional membranes and was intermittently observed in the sinus endothelium. These data suggest that adherens junctions, whose formation appears to be mediated by VE-cadherin-catenin complexes, probably regulate the passage of blood cells through the spleen. This work was supported by a Grant-in-Aid for Scientific Research (C), Japan     

Combined administration of G-CSF and GM-CSF stimulates monocyte-derived pro-angiogenic cells in patients with acute myocardial infarction

Mobilization of endothelial progenitor cells has been suggested to contribute to neo-vascularization of ischemic organs. Aim of this study was to investigate whether the combination of granulocyte colony stimulating factor (G-CSF) and granulocyte-macrophage (GM)-CSF may influence the expansion of circulating KDR+ cells in patients with acute myocardial infarction (AMI). KDR+ cells significantly increased in peripheral blood of AMI patients treated with G-CSF and GM-CSF compared to untreated patients. This KDR+ cells population was CD14+ but not CD34+ or CD133+. CD14+/KDR+ cells were also obtained in vitro by culturing mononuclear cells from healthy donors in a Rotary Cell Culture System in the presence of G-CSF + GM-CSF, but not of the individual growth factors. CD14+/KDR+ cells, obtained from patients or from in vitro culture, co-expressed hematopoietic (CD45, CD14) and endothelial markers (CD31, CD105, and VE-cadherin). CD14+/KDR+, but not CD14+/KDR- cells, stimulated the organization of human microvascular endothelial cells into capillary-like structures on Matrigel both in vitro and in vivo. The combination of G-CSF and GM-CSF induced a CD14+/KDR+ cell population with potential pro-angiogenic properties.     

Cleavage of β-Catenin and Plakoglobin and Shedding of VE-Cadherin during Endothelial Apoptosis: Evidence for a Role for Caspases and Metalloproteinases

Growth factor deprivation of endothelial cells induces apoptosis, which is characterized by membrane blebbing, cell rounding, and subsequent loss of cell–matrix and cell–cell contacts. In this study, we show that initiation of endothelial apoptosis correlates with cleavage and disassembly of intracellular and extracellular components of adherens junctions. β-Catenin and plakoglobin, which form intracellular links between vascular endothelial cadherin (VE-cadherin) and actin-binding α-catenin in adherens junctions, are cleaved in apoptotic cells. In vitro incubations of cell lysates and immunoprecipitates with recombinant caspases indicate that CPP32 and Mch2 are involved, possibly by initiating proteolytic processing. Cleaved β-catenin from lysates of apoptotic cells does not bind to endogenous α-catenin, whereas plakoglobin retains its binding capacity. The extracellular portion of the adherens junctions is also altered during apoptosis because VE-cadherin, which mediates endothelial cell–cell interactions, dramatically decreases on the surface of cells. An extracellular fragment of VE-cadherin can be detected in the conditioned medium, and this “shedding” of VE-cadherin can be blocked by an inhibitor of metalloproteinases. Thus, cleavage of β-catenin and plakoglobin and shedding of VE-cadherin may act in concert to disrupt structural and signaling properties of adherens junctions and may actively interrupt extracellular signals required for endothelial cell survival.     

Vinculin associates with endothelial VE-cadherin junctions to control force-dependent remodeling

To remodel endothelial cell-cell adhesion, inflammatory cytokine- and angiogenic growth factor-induced signals impinge on the vascular endothelial cadherin (VE-cadherin) complex, the central component of endothelial adherens junctions. This study demonstrates that junction remodeling takes place at a molecularly and phenotypically distinct subset of VE-cadherin adhesions, defined here as focal adherens junctions (FAJs). FAJs are attached to radial F-actin bundles and marked by the mechanosensory protein Vinculin. We show that endothelial hormones vascular endothelial growth factor, tumor necrosis factor α, and most prominently thrombin induced the transformation of stable junctions into FAJs. The actin cytoskeleton generated pulling forces specifically on FAJs, and inhibition of Rho-Rock-actomyosin contractility prevented the formation of FAJs and junction remodeling. FAJs formed normally in cells expressing a Vinculin binding-deficient mutant of α-catenin, showing that Vinculin recruitment is not required for adherens junction formation. Comparing Vinculin-devoid FAJs to wild-type FAJs revealed that Vinculin protects VE-cadherin junctions from opening during their force-dependent remodeling. These findings implicate Vinculin-dependent cadherin mechanosensing in endothelial processes such as leukocyte extravasation and angiogenesis.     

Isolation of cardiac cells from E8.5 yolk sac by ALCAM (CD166) expression

It is known that the adhesion molecule ALCAM (CD166) mediates metastasis of malignant cells and organogenesis in embryos. We show here that embryonic day 8.5 (E8.5) murine yolk sac cells express ALCAM protein and that ALCAM expression can be used to define endothelial and cardiac precursors from hematopoietic precursors in E8.5 yolk sacs. ALCAM high+ cells exclusively give rise to endothelial and cardiac cells in matrigel assays but generate no hematopoietic colonies in methylcellulose assays. ALCAM low+ and ALCAM- populations predominantly give rise to hematopoietic cells in methylcellulose, but do not generate any cell clusters in matrigel. The ALCAM high+ population contains both Flk-1+ and Flk-1- cells. The former population exclusively contains endothelial cells whereas the latter give rise to cardiac cells when cultured on OP9 stromal cells. We also show that cardiac lineage marker genes such as Nkx-2.5, and the endothelial marker VE-cadherin are expressed in the ALCAM high+ fraction, whereas the hematopoietic marker GATA1 and Runx1 are expressed in the ALCAM low+/- fraction. However, we did not detect expression of the cardiac structural protein cTn-T in cells from yolk sac cells until these had had been differentiated on OP9 for 5 days. Altogether, these results indicate that cells retaining a potential to differentiate to the cardiac lineage are present in E8.5 yolk sacs and can be isolated as ALCAM high+, Flk-1- cells. Our report provides novel insights into the origin and differentiation process of cardiac cells in the formation of the circulatory system.     

SHP2 association with VE-cadherin complexes in human endothelial cells is regulated by thrombin

Thrombin-mediated changes in endothelial cell adherens junctions modulate vascular permeability. We demonstrate that the nonreceptor protein-tyrosine phosphatase SHP2 co-precipitates with VE-cadherin complexes in confluent, quiescent human umbilical vein endothelial cells. Ligand-binding blots using a SHP2-glutathione S-transferase fusion peptide established that SHP2 associates selectively with beta-catenin in VE-cadherin complexes. Thrombin treatment of human umbilical vein endothelial cells promotes SHP2 tyrosine phosphorylation and dissociation from VE-cadherin complexes. The loss of SHP2 from the cadherin complexes correlates with a dramatic increase in the tyrosine phosphorylation of beta-catenin, gamma-catenin, and p120-catenin complexed with VE-cadherin. We propose that thrombin regulates the tyrosine phosphorylation of VE-cadherin-associated beta-catenin, gamma-catenin, and p120-catenin by modulating the quantity of SHP2 associated with VE-cadherin complexes. Such changes in adherens junction complex composition likely underlie thrombin-elicited alterations in endothelial monolayer permeability.     

The molecular organization of endothelial junctions and their functional role in vascular morphogenesis and permeability

We review here our work on the molecular and functional organization of endothelial cell-to-cell junctions. The first part of the review is dedicated to VE-cadherin, characterized by our group few years ago. This protein is a member of the large family of transmembrane adhesion proteins called cadherins. It is endothelial cell specific and plays a major role in the organization of adherens junctions. Inactivation of VE-cadherin gene or in vivo truncation of its cytoplasmic tail leads to a lethal phenotype due to the lack of correct organization of the vasculature in the embryo. We found that the defect was due to apoptosis of endothelial cells, which became unresponsive to the survival signal induced by vascular endothelial cell growth factor. Our data indicate that VE-cadherin may act as a scaffolding protein able to associate vascular endothelial cell growth factor receptor and to promote its signaling. In the second part of the review we consider another protein more recently discovered by us and called junctional adhesion molecule (JAM). This protein is a small immunoglobulin which is located at tight junctions in the endothelium and in epithelial cells. Evidence is discussed indicating that JAM takes part in the organization of tight junctions and modulates leukocyte extravasation through endothelial intercellular junctions in vitro and in vivo. The general role of tight junctions in endothelial cells is also discussed.     

Endothelial cells within embryonic skeletal muscles: a potential source of myogenic progenitors

We investigated whether the vessel-associated or endothelial cells within mouse embryo muscles can be a source of myogenic progenitors. Immunodetection of the stem cell surface markers, CD34 and Flk1, which are known to characterize the endothelial lineage, was done throughout the course of embryo muscle development. Both markers appeared to be restricted to the vessel-associated cells. On the basis of CD34 labeling, the reactive cells were purified by magnetic-bead selection from the limb muscles of 17-dpc desmin+/-LacZ mouse embryos and characterized by fluorescence-activated cell sorting. The cells in the selected CD34(+) population appeared to be approximately 95% positive for Flk1, but usually negative for CD45. We demonstrated that in vitro the CD34(+)/Flk1(+) population differentiated into endothelial cells and skeletal myofibers. When transplanted into mdx mouse muscle, this population displayed a high propensity to disperse within the recipient muscle, fuse with the host myofibers, and restore dystrophin expression. The marked ability of the embryonic muscle endothelial cells to activate myogenic program could be related to their somitic origin.     

Beyond vessels: occurrence and regional clustering of vascular endothelial (VE-)cadherin-containing junctions in non-endothelial cells

The genes encoding transmembrane glycoproteins of the cadherin family, i.e., the Ca²⁺-dependent cell-cell adhesion molecules, are typically expressed in cell-type- or cell-lineage-specific patterns. One of them, vascular endothelial (VE)-cadherin, is widely considered to be specific for vascular endothelia in which it is either the sole or the predominant cadherin, often co-existing with N-cadherin. This specificity of VE-cadherin for vascular endothelial cells is important not only in blood and lymph vessel biology and medicine, but also for cell-type-based diagnoses, notably those of metastatic tumors. Surprisingly, however, we have recently noted the frequent synthesis, surface exposure, and junction assembly of VE-cadherin in certain other cells, in which this glycoprotein is clustered into adherens junctions (AJs), either alone or in combination with N-cadherin and/or cadherin-11. Such cells include mammalian astrocytes and glioma, probably mostly astrocytoma cells growing in culture, and a specific subtype of astrocytoma in situ. Moreover, VE-cadherin synthesis and AJ assembly, plus the regional clustering of such AJs in certain domains, are not clonally fixed but can appear again and again in cells of the progeny of cloned homogeneous-appearing individual cells, thus resulting in clonal cell colonies that are often heterogeneous in their cadherin junction patterns. We discuss the constitutive presence of VE-cadherin in some non-endothelial cells with respect to certain architectural features and possible physiological and pathogenic functions of the cells, and in comparison with recent reports of VE-cadherin-positive melanomas. This work was supported in part by the Deutsche Krebshilfe (grant 10 2049 Fr1) and the German Ministry for Research and Technology (Program Regenerative Medicine, START-MSC consortium).     

Decreased expression of VE-cadherin and claudin-5 and increased phosphorylation of VE-cadherin in vascular endothelium in nasal polyps

VE-cadherin and claudin-5 are major components of adherens and tight junctions of vascular endothelial cells and a decrease in their expression and an increase in the tyrosine-phosphorylation of VE-cadherin are associated with an increase in endothelial paracellular permeability. To clarify the mechanism underlying the development of edema in nasal polyps, we studied these molecules in polyp microvessels. Normal inferior turbinate mucosal tissues and nasal polyps from patients treated with or without glucocorticoid were stained for VE-cadherin or claudin-5 and CD31 by a double-immunofluorescence method and the immunofluorescence intensities were graded 1–3 with increasing intensity. To correct for differences in fluorescence intensity attributable to a different endothelial area being exposed in a section or to the thickness of a section, the relative immunofluorescence intensity was estimated by dividing the grade of VE-cadherin or claudin-5 by that of CD31 in each microvessel. Tyrosine-phosphorylation of VE-cadherin was examined by Western blot analysis. The relative intensities of VE-cadherin and claudin-5 in the CD31-positive microvessels significantly decreased in the following order; inferior turbinate mucosa, treated polyps and untreated polyps. The ratio of tyrosine-phosphorylated VE-cadherin to VE-cadherin was significantly higher in untreated polyps than in the inferior turbinate mucosa and treated polyps, between which no significant difference in the ratio was seen. Thus, in nasal polyps, the barrier function of endothelial adherens and tight junctions is weakened, although glucocorticoid treatment improves this weakened barrier function.     

Unraveling the distinct distributions of VE- and N-cadherins in endothelial cells: A key role for p120-catenin

Endothelial cells express two different classical cadherins, vascular endothelial (VE) cadherin and neural (N) cadherin, having distinct functions in the vascular system. VE-cadherin is specific to endothelial adherens junctions and is strictly necessary for vascular morphogenesis. On the contrary, N-cadherin shows diffuse localization on the cell surface and interacts with mural cells for vessel stabilization. In this study, we sought to clarify the cellular mechanisms leading to the distinct cellular locations and functions of the two cadherins in the endothelium. VE-cadherin has been shown to be responsible for the junctional exclusion of N-cadherin. Using several endothelial models, we demonstrate that this property is dependent on VE-cadherin binding to p120 catenin (p120^ctn). Moreover, although in the absence of VE-cadherin N-cadherin can localize to cell contacts, angiogenesis remains impaired, demonstrating that endothelial junction formation is not sufficient for normal vessel development. Interestingly, we show that VE-cadherin, but not N-cadherin, is partially associated with cholesterol-enriched microdomains. Lipid raft-associated-VE-cadherin is characterized by a very high level of p120^ctn association, and this association is necessary for VE-cadherin recruitment into lipid rafts. Altogether, our results indicate a critical role for p120^ctn in regulating the membrane distribution of endothelial cadherins with functional consequences in terms of cadherin stabilization and intracellular signaling.     

Activation of MMP-2, cleavage of matrix proteins,and adherens junctions during a snake venom metalloproteinase-induced endothelial cell apoptosis

Snake venom metalloproteinases (SVMPs) are structurally and functionally similar to matrix metalloproteinases (MMPs). We have previously demonstrated that a SVMP, named gaminelysin, can induce endothelial cell apoptosis [Biochem J. 357 (2001) 719]. In this study, the action mechanism of graminelysin in causing endothelial cell apoptosis was further investigated. We showed that the apoptosis was initiated with cell shape change and extracellular matrix degradation and occurred before cell detachment. Cleaved forms of MMP-2 might act in concert with graminelysin to cause apoptosis. During apoptosis, adherens junctions, including VE-cadherin and beta- and gamma-catenin were cleaved and alpha-catenin was decreased. VE-cadherin and beta-catenin at cell periphery were decreased and the discontinuity in alignment was found as observed with immunofluorescence microscopy. This was accompanied with a diffuse beta-catenin staining in the cytoplasm and a decreased F-actin stress fibers in some rounded cells. The decrease of VE-cadherin and beta-catenin in Triton-insoluble fractions confirmed that the association of adherens junctions with actin cytoskeleton was altered during apoptosis. Graminelysin-induced cleavage in adherens junctions was paralleled with the changes in paracellular permeability. We also detected the activation of caspase-3 and the decrease of Bcl-2/Bax ratio during apoptosis. However, caspase inhibitors showed differential effects in blocking the cleavage of PARP, adherens junctions, and DNA fragmentation. Taken together, the data presented suggest that metalloproteinase can control cell fates via the degradation of matrix proteins, the change of cell shape, and the cleavage of adherens junctions.