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.

A Splice-Isoform of Vesicle-associated Membrane Protein-1 (VAMP-1) Contains a Mitochondrial Targeting Signal

Screening of a library derived from primary human endothelial cells revealed a novel human isoform of vesicle-associated membrane protein-1 (VAMP-1), a protein involved in the targeting and/or fusion of transport vesicles to their target membrane. We have termed this novel isoform VAMP-1B and designated the previously described isoform VAMP-1A. VAMP-1B appears to be an alternatively spliced form of VAMP-1. A similar rat splice variant of VAMP-1 (also termed VAMP-1B) has recently been reported. Five different cultured cell lines, from different lineages, all contained VAMP-1B but little or no detectable VAMP-1A mRNA, as assessed by PCR. In contrast, brain mRNA contained VAMP-1A but no VAMP-1B. The VAMP-1B sequence encodes a protein identical to VAMP-1A except for the carboxy-terminal five amino acids. VAMP-1 is anchored in the vesicle membrane by a carboxy-terminal hydrophobic sequence. In VAMP-1A the hydrophobic anchor is followed by a single threonine, which is the carboxy-terminal amino acid. In VAMP-1B the predicted hydrophobic membrane anchor is shortened by four amino acids, and the hydrophobic sequence is immediately followed by three charged amino acids, arginine-arginine-aspartic acid. Transfection of human endothelial cells with epitope-tagged VAMP-1B demonstrated that VAMP-1B was targeted to mitochondria whereas VAMP-1A was localized to the plasma membrane and endosome-like structures. Analysis of C-terminal mutations of VAMP-1B demonstrated that mitochondrial targeting depends both on the addition of positive charge at the C terminus and a shortened hydrophobic membrane anchor. These data suggest that mitochondria may be integrated, at least at a mechanistic level, to the vesicular trafficking pathways that govern protein movement between other organelles of the cell.

The Fibrinogen Globe of Tenascin-C Promotes Basic Fibroblast Growth Factor-induced Endothelial Cell Elongation

To investigate the potential role of tenascin-C (TN-C) on endothelial sprouting we used bovine aortic endothelial cells (BAECs) as an in vitro model of angiogenesis. We found that TN-C is specifically expressed by sprouting and cord-forming BAECs but not by nonsprouting BAECs. To test whether TN-C alone or in combination with basic fibroblast growth factor (bFGF) can enhance endothelial sprouting or cord formation, we used BAECs that normally do not sprout and, fittingly, do not express TN-C. In the presence of bFGF, exogenous TN-C but not fibronectin induced an elongated phenotype in nonsprouting BAECs. This phenotype was due to altered actin cytoskeleton organization. The fibrinogen globe of the TN-C molecule was the active domain promoting the elongated phenotype in response to bFGF. Furthermore, we found that the fibrinogen globe was responsible for reduced cell adhesion of BAECs on TN-C substrates. We conclude that bFGF-stimulated endothelial cells can be switched to a sprouting phenotype by the decreased adhesive strength of TN-C, mediated by the fibrinogen globe.

Time-dependent vascular regression and permeability changes in established human tumor xenografts induced by an anti-vascular endothelial growth factor/vascular permeability factor antibody

The hyperpermeability of tumor vessels to macromolecules, compared with normal vessels, is presumably due to vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) released by neoplastic and/or host cells. In addition, VEGF/VPF is a potent angiogenic factor. Removal of this growth factor may reduce the permeability and inhibit tumor angiogenesis. To test these hypotheses, we transplanted a human glioblastoma (U87), a human colon adenocarcinoma (LS174T), and a human melanoma (P-MEL) into two locations in immunodeficient mice: the cranial window and the dorsal skinfold chamber. The mice bearing vascularized tumors were treated with a bolus (0.2 ml) of either a neutralizing antibody (A4.6.1) (492 μg/ml) against VEGF/VPF or PBS (control). We found that tumor vascular permeability to albumin in antibody-treated groups was lower than in the matched controls and that the effect of the antibody was time-dependent and influenced by the mode of injection. Tumor vascular permeability did not respond to i.p. injection of the antibody until 4 days posttreatment. However, the permeability was reduced within 6 h after i.v. injection of the same amount of antibody. In addition to the reduction in vascular permeability, the tumor vessels became smaller in diameter and less tortuous after antibody injections and eventually disappeared from the surface after four consecutive treatments in U87 tumors. These results demonstrate that tumor vascular permeability can be reduced by neutralization of endogenous VEGF/VPF and suggest that angiogenesis and the maintenance of integrity of tumor vessels require the presence of VEGF/VPF in the tissue microenvironment. The latter finding reveals a new mechanism of tumor vessel regression—i.e., blocking the interactions between VEGF/VPF and endothelial cells or inhibiting VEGF/VPF synthesis in solid tumors causes dramatic reduction in vessel diameter, which may block the passage of blood elements and thus lead to vascular regression.

Streptavidin facilitates internalization and pulmonary targeting of an anti-endothelial cell antibody (platelet-endothelial cell adhesion molecule 1): A strategy for vascular immunotargeting of drugs

Conjugation of drugs with antibodies to surface endothelial antigens is a potential strategy for drug delivery to endothelium. We studied antibodies to platelet-endothelial adhesion molecule 1 (PECAM-1, a stably expressed endothelial antigen) as carriers for vascular immunotargeting. Although 125I-labeled anti-PECAM bound to endothelial cells in culture, the antibody was poorly internalized by the cells and accumulated poorly after intravenous administration in mice and rats. However, conjugation of biotinylated anti-PECAM (b-anti-PECAM) with streptavidin (SA) markedly stimulated uptake and internalization of anti-PECAM by endothelial cells and by cells expressing PECAM. In addition, conjugation with streptavidin markedly stimulated uptake of 125I-labeled b-anti-PECAM in perfused rat lungs and in the lungs of intact animals after either intravenous or intraarterial injection. The antioxidant enzyme catalase conjugated with b-anti-PECAM/SA bound to endothelial cells in culture, entered the cells, escaped intracellular degradation, and protected the cells against H2O2-induced injury. Anti-PECAM/SA/125I-catalase accumulated in the lungs after intravenous injection or in the perfused rat lungs and protected these lungs against H2O2-induced injury. Thus, modification of a poor carrier antibody with biotin and SA provides an approach for facilitation of antibody-mediated drug targeting. Anti-PECAM/SA is a promising candidate for vascular immunotargeting of bioactive drugs.

Angiostatin gene transfer: Inhibition of tumor growth in vivo by blockage of endothelial cell proliferation associated with a mitosis arrest

The antitumoral effects that follow the local delivery of the N-terminal fragment of human plasminogen (angiostatin K3) have been studied in two xenograft murine models. Angiostatin delivery was achieved by a defective adenovirus expressing a secretable angiostatin K3 molecule from the cytomegalovirus promoter (AdK3). In in vitro studies, AdK3 selectively inhibited endothelial cell proliferation and disrupted the G2/M transition induced by M-phase-promoting factors. AdK3-infected endothelial cells showed a marked mitosis arrest that correlated with the down-regulation of the M-phase phosphoproteins. A single intratumoral injection of AdK3 into preestablished rat C6 glioma or human MDA-MB-231 breast carcinoma grown in athymic mice was followed by a significant arrest of tumor growth, which was associated with a suppression of neovascularization within and at the vicinity of the tumors. AdK3 therapy also induced a 10-fold increase in apoptotic tumor cells as compared with a control adenovirus. Furthermore, we showed that systemic injection of AdK3 delayed C6 tumor establishment and growth, confirming that angiostatin can function in a paracrin manner. Our data support the concept that targeted antiangiogenesis, using adenovirus-mediated gene transfer, represents a promising alternative strategy for delivering antiangiogenic factors as their bolus injections present unsolved pharmacological problems.

Endothelial cell death, angiogenesis, and microvascular function after castration in an androgen-dependent tumor: Role of vascular endothelial growth factor

The sequence of events that leads to tumor vessel regression and the functional characteristics of these vessels during hormone–ablation therapy are not known. This is because of the lack of an appropriate animal model and monitoring technology. By using in vivo microscopy and in situ molecular analysis of the androgen-dependent Shionogi carcinoma grown in severe combined immunodeficient mice, we show that castration of these mice leads to tumor regression and a concomitant decrease in vascular endothelial growth factor (VEGF) expression. Androgen withdrawal is known to induce apoptosis in Shionogi tumor cells. Surprisingly, tumor endothelial cells begin to undergo apoptosis before neoplastic cells, and rarefaction of tumor vessels precedes the decrease in tumor size. The regressing vessels begin to exhibit normal phenotype, i.e., lower diameter, tortuosity, vascular permeability, and leukocyte adhesion. Two weeks after castration, a second wave of angiogenesis and tumor growth begins with a concomitant increase in VEGF expression. Because human tumors often relapse following hormone–ablation therapy, our data suggest that these patients may benefit from combined anti-VEGF therapy.

Nuclear localization of prostaglandin E2 receptors

Prostaglandin E2 receptors (EP) were detected by radioligand binding in nuclear fractions isolated from porcine brain and myometrium. Intracellular localization by immunocytofluorescence revealed perinuclear localization of EPs in porcine cerebral microvascular endothelial cells. Nuclear association of EP1 was also found in fibroblast Swiss 3T3 cells stably overexpressing EP1 and in human embryonic kidney 293 (Epstein–Barr virus-encoded nuclear antigen) cells expressing EP1 fused to green fluorescent protein. High-resolution immunostaining of EP1 revealed their presence in the nuclear envelope of isolated (cultured) endothelial cells and in situ in brain (cortex) endothelial cells and neurons. Stimulation of these nuclear receptors modulate nuclear calcium and gene transcription.

Inhibition of Endothelial Wound Repair by Dominant Negative Connexin Inhibitors

Wounding of endothelial cells is associated with altered direct intercellular communication. To determine whether gap junctional communication participates to the wound repair process, we have compared connexin (Cx) expression, cell-to-cell coupling and kinetics of wound repair in monolayer cultures of PymT-transformed mouse endothelial cells (clone bEnd.3) and in bEnd.3 cells expressing different dominant negative Cx inhibitors. In parental bEnd.3 cells, mechanical wounding increased expression of Cx43 and decreased expression of Cx37 at the site of injury, whereas Cx40 expression was unaffected. These wound-induced changes in Cx expression were associated with functional changes in cell-to-cell coupling, as assessed with different fluorescent tracers. Stable transfection with cDNAs encoding for the chimeric connexin 3243H7 or the fusion protein Cx43-βGal resulted in perturbed gap junctional communication between bEnd.3 cells under both basal and wounded conditions. The time required for complete repair of a defined wound within a confluent monolayer was increased by ∼50% in cells expressing the dominant negative Cx inhibitors, whereas other cell properties, such as proliferation rate, migration of single cells, cyst formation and extracellular proteolytic activity, were unaltered. These findings demonstrate that proper Cx expression is required for coordinated migration during repair of an endothelial wound.

Endothelial Transcytotic Machinery Involves Supramolecular Protein–Lipid Complexes

We have demonstrated that the plasmalemmal vesicles (caveolae) of the continuous microvascular endothelium function as transcytotic vesicular carriers for protein molecules >20 Å and that transcytosis is an N-ethylmaleimide–sensitive factor (NSF)-dependent, N-ethylmaleimide-sensitive process. We have further investigated NSF interactions with endothelial proteins to find out 1) whether a complete set of fusion and targeting proteins is present in the endothelium; 2) whether they are organized in multimolecular complexes as in neurons; and 3) whether the endothelial multimolecular complexes differ from their neuronal counterparts, because of their specialized role in transcytosis. To generate the complexes, we have used myc-NSF, cultured pulmonary endothelial cells, and rat lung cytosol and membrane preparations; to detect them we have applied coimmunoprecipitation with myc antibodies; and to characterize them we have used velocity sedimentation and cross-linking procedures. We have found that both cytosolic and membrane fractions contain complexes that comprise beside soluble NSF attachment proteins and SNAREs (soluble NSF attachment protein receptor), rab 5, dynamin, caveolin, and lipids. By immunogold labeling and negative staining we have detected in these complexes, myc-NSF, syntaxin, dynamin, caveolin, and endogenous NSF. Similar complexes are formed by endogenous NSF. The results indicate that complexes with a distinct protein–lipid composition exist and suggest that they participate in targeting, fusion, and fission of caveolae with the endothelial plasmalemma.

Platelets modulate gastric ulcer healing: Role of endostatin and vascular endothelial growth factor release

Bleeding and delayed healing of ulcers are well recognized clinical problems associated with the use of aspirin and other nonsteroidal antiinflammatory drugs, which have been attributed to their antiaggregatory effects on platelets. We hypothesized that antiplatelet drugs might interfere with gastric ulcer healing by suppressing the release of growth factors, such as vascular endothelial growth factor (VEGF), from platelets. Gastric ulcers were induced in rats by serosal application of acetic acid. Daily oral treatment with vehicle, aspirin, or ticlopidine (an ADP receptor antagonist) was started 3 days later and continued for 1 week. Ulcer induction resulted in a significant increase in serum levels of VEGF and a significant decrease in serum levels of endostatin (an antiangiogenic factor). Although both aspirin and ticlopidine markedly suppressed platelet aggregation, only ticlopidine impaired gastric ulcer healing and angiogenesis as well as reversing the ulcer-associated changes in serum levels of VEGF and endostatin. The effects of ticlopidine on ulcer healing and angiogenesis were mimicked by immunodepletion of circulating platelets, and ticlopidine did not influence ulcer healing when given to thrombocytopenic rats. Incubation of human umbilical vein endothelial cells with serum from ticlopidine-treated rats significantly reduced proliferation and increased apoptosis, effects reversed by an antibody directed against endostatin. Ticlopidine treatment resulted in increased platelet endostatin content and release. These results demonstrate a previously unrecognized contribution of platelets to the regulation of gastric ulcer healing. Such effects likely are mediated through the release from platelets of endostatin and possibly VEGF. As shown with ticlopidine, drugs that influence gastric ulcer healing may do so in part through altering the ability of platelets to release growth factors.

How the protease thrombin talks to cells

How does a protease act like a hormone to regulate cellular functions? The coagulation protease thrombin (EC 3.4.21.5) activates platelets and regulates the behavior of other cells by means of G protein-coupled protease-activated receptors (PARs). PAR1 is activated when thrombin binds to and cleaves its amino-terminal exodomain to unmask a new receptor amino terminus. This new amino terminus then serves as a tethered peptide ligand, binding intramolecularly to the body of the receptor to effect transmembrane signaling. The irreversibility of PAR1’s proteolytic activation mechanism stands in contrast to the reversible ligand binding that activates classical G protein-coupled receptors and compels special mechanisms for desensitization and resensitization. In endothelial cells and fibroblasts, activated PAR1 rapidly internalizes and then sorts to lysosomes rather than recycling to the plasma membrane as do classical G protein-coupled receptors. This trafficking behavior is critical for termination of thrombin signaling. An intracellular pool of thrombin receptors refreshes the cell surface with naïve receptors, thereby maintaining thrombin responsiveness. Thus cells have evolved a trafficking solution to the signaling problem presented by PARs. Four PARs have now been identified. PAR1, PAR3, and PAR4 can all be activated by thrombin. PAR2 is activated by trypsin and by trypsin-like proteases but not by thrombin. Recent studies with knockout mice, receptor-activating peptides, and blocking antibodies are beginning to define the role of these receptors in vivo.

Endothelial cell surface F1-FO ATP synthase is active in ATP synthesis and is inhibited by angiostatin

Angiostatin blocks tumor angiogenesis in vivo, almost certainly through its demonstrated ability to block endothelial cell migration and proliferation. Although the mechanism of angiostatin action remains unknown, identification of F1-FO ATP synthase as the major angiostatin-binding site on the endothelial cell surface suggests that ATP metabolism may play a role in the angiostatin response. Previous studies noting the presence of F1 ATP synthase subunits on endothelial cells and certain cancer cells did not determine whether this enzyme was functional in ATP synthesis. We now demonstrate that all components of the F1 ATP synthase catalytic core are present on the endothelial cell surface, where they colocalize into discrete punctate structures. The surface-associated enzyme is active in ATP synthesis as shown by dual-label TLC and bioluminescence assays. Both ATP synthase and ATPase activities of the enzyme are inhibited by angiostatin as well as by antibodies directed against the α- and β-subunits of ATP synthase in cell-based and biochemical assays. Our data suggest that angiostatin inhibits vascularization by suppression of endothelial-surface ATP metabolism, which, in turn, may regulate vascular physiology by established mechanisms. We now have shown that antibodies directed against subunits of ATP synthase exhibit endothelial cell-inhibitory activities comparable to that of angiostatin, indicating that these antibodies function as angiostatin mimetics.

A mouse model for adenovirus gene delivery

The cellular attachment receptor for adenovirus (Ad), Coxsackie adenovirus receptor (CAR), required for delivery of Ad into primary cells, is not present on all cell types, thus restricting Ad-gene delivery systems. To circumvent this constrain, a transgenic mouse has been generated that expresses a truncated human CAR in all tissues analyzed. These mice allowed efficient in vitro infections at low multiplicities into lymphoid, myeloid, and endothelial cells. Furthermore, in vivo administration of Ad-vectors results in infection of macrophages, lymphocytes, and endothelial cells. In addition, tail vein injection resulted in targeting of virus into previously inaccessible areas, such as the lung and the capillaries of the brain. The CAR transgenic mice will be useful for rapid functional genomic analysis in vivo, for testing the efficacy of gene therapy procedures or as a source of easily transducible cells.

Expression and functional significance of VE-cadherin in aggressive human melanoma cells: Role in vasculogenic mimicry

We recently have introduced the term vasculogenic mimicry to describe the unique ability of aggressive melanoma tumor cells to form tubular structures and patterned networks in three-dimensional culture, which “mimics” embryonic vasculogenic networks formed by differentiating endothelial cells. In the current study, we address the biological significance of several endothelial-associated molecules (revealed by microarray analysis) with respect to expression and function in highly aggressive and poorly aggressive human cutaneous melanoma cell lines (established from the same patient). In a comparative analysis, CD31 was not expressed by any of the melanoma cell lines, whereas TIE-1 (tyrosine kinase with Ig and epidermal growth factor homology domains-1) was strongly expressed in the highly aggressive tumor cells with a low level of expression in one of the poorly aggressive cell lines. Vascular endothelial (VE)-cadherin was exclusively expressed by highly aggressive melanoma cells and was undetectable in the poorly aggressive tumor cells, suggesting the possibility of a vasculogenic switch. Down-regulation of VE-cadherin expression in the aggressive melanoma cells abrogated their ability to form vasculogenic networks and directly tested the hypothesis that VE-cadherin is critical in melanoma vasculogenic mimicry. These results highlight the plasticity of aggressive melanoma cells and call into question their possible genetic reversion to an embryonic phenotype. This finding could pose a significant clinical challenge in targeting tumor cells that may masquerade as circulating endothelial cells or other embryonic-like stem cells.