Thioredoxin inhibits microvascular endothelial capillary tubule formation

Thioredoxin (Trx) inhibited human HMEC-1 dermal microvascular endothelial cell capillary tubule forming capacity in a Matrigel based assay in vitro. Inhibition of capillary tubule formation was Trx catalytic site and thioredoxin reductase (TrxR) dependent, mediated at the Matrigel matrix level, and associated with a shift from morphological differentiation to continuous proliferation, with enhanced cell spreading resulting in eventual monolayer formation. Soluble complex carbohydrates, which inhibited capillary tubule formation on Matrigel without induction of cell spreading or monolayer formation, failed to impair Trx promotion of cell spreading and mono-layer formation, suggesting a shift away from carbohydrate-mediated cell/matrix adhesive interactions. Laminin peptides YIGRS and SIKVAV, which impaired tubule formation on Matrigel without inducing cell spreading or monolayer formation, partially impaired cell spreading upon Trx-treated Matrigel without restoring tubule formation, consistent with a potential role for laminin in Trx-mediated effects. Trx reduced laminin and destabilised laminin/galectin-3 complexes within Matrigel. Native purified EHS Laminin (also containing galectin-3), but not recombinant galectin-3, restored HMEC-1 capillary tubule formation on Trx-treated Matrigel. These data highlight a novel deregulatory effect of extracellular Trx upon morphological capillary differentiation that appears to depend upon the reduction of laminin and destabilisation of its interaction with galectin-3, possibly leading to galectin-3 neutralisation that shifts cell/matrix adhesive interactions away from being carbohydrate mediated and results in loss of proliferation-inhibiting and differentiation promoting cues from this tumor basement membrane matrix.     

DNAzymes to beta 1 and beta 3 mRNA down-regulate expression of the targeted integrins and inhibit endothelial cell capillary tube formation in fibrin and matrigel.

A novel approach based on DNA-cleaving deoxyribozymes (DNAzymes) was developed to control expression of beta(1) and beta(3) integrins in endothelial cells. To engineer a specific cleavage site in mRNA, the flanking domains of DNAzymes were derived from oligodeoxynucleotides complementary to sequences corresponding to 1053-1070 and 1243-1267 in beta(1) and beta(3) mRNA, respectively. Phosphorothioate analogues of these antisense oligodeoxynucleotides, designated beta1-1053 and beta3-1243, significantly inhibited expression of beta(1) and beta(3) integrin subunits in endothelial and K562 cells at the level of mRNA and protein synthesis. They also specifically decreased the cell surface expression of corresponding subunits in endothelial cells and K562 cells, as measured by flow cytometry. In functional tests, beta1-1053 and beta3-1243 markedly reduced adhesion of cells to fibronectin and vitronectin, respectively. We designed DNAzymes to beta(1) and beta(3) mRNAs containing a 15-deoxynucleotide catalytic domain that was flanked by two substrate recognition segments of 8 and 10 deoxynucleotides for beta(1) and beta(3) DNAzymes, respectively. Both DNAzymes in the presence of Mg(2+) specifically cleaved their substrates, synthetic beta(1) and beta(3) mRNA fragments. Although DNAzymes were partially modified with phosphorothioate and with 2'-O-methyl groups at both the 5' and 3' ends indicated similar kinetic parameters, they were significantly more potent than the unmodified DNAzymes because of their much higher resistance to nuclease degradation. Similar to the antisense oligonucleotides, DNAzymes abolished microvascular endothelial cell capillary tube formation in fibrin and Matrigel. In conclusion, DNAzymes to beta(1) and beta(3) mRNAs with 2'-O-methyl modifications are potentially useful as gene-inactivating agents and may ultimately provide a therapeutic means to inhibit angiogenesis in vivo.     

Fibrin II induces endothelial cell capillary tube formation

We studied the formation of capillary tubes by endothelial cells which were sandwiched between two fibrin gels under serum-free conditions. After formation of the overlying fibrin gel, the endothelial cell monolayer rearranged into an extensive net of capillary tubes. Tube formation was apparent at 5 h and was fully developed by 24 h. The capillary tubes were vacuolated, and both intracellular and intercellular lumina were present. Maximal tube formation was observed with fibrin II (which lacks both fibrinopeptide A and B), minimal tube formation with fibrin I (which lacks only fibrinopeptide A), and complete absence of tube formation with fibrin 325 (which lacks the NH2- terminal beta 15-42 sequence, in addition to fibrinopeptides A and B). The inability of fibrin 325 to stimulate capillary tube formation supports the idea that beta 15-42 plays an important role in this process, and its importance was confirmed by the finding that exogenous soluble beta 15-42 inhibited fibrin II-induced capillary tube formation. This effect was specific for fibrin, since beta 15-42 did not inhibit tube formation by endothelial cells sandwiched between collagen gels. The interaction of the apical surface of the endothelial cell with the overlying fibrin II gel, as opposed to the underlying fibrin gel upon which the cells were seeded, was necessary for capillary tube formation. These studies suggest that the beta 15-42 sequence of fibrin interacts with a component of the apical cell surface and that this interaction plays a fundamental role in the induction of endothelial capillary tube formation.     

Human microvascular endothelial cells use beta 1 and beta 3 integrin receptor complexes to attach to laminin

Microvascular endothelial cells (MEC) use a set of surface receptors to adhere not only to the vascular basement membrane but, during angiogenic stimulation, to the interstitium. We examined how cultured human MEC interact with laminin-rich basement membranes. By using a panel of monoclonal antibodies, we found that MEC cells express a number of integrin-related receptor complexes, including alpha 1 beta 1, alpha 2 beta 1, alpha 3 beta 1, alpha 5 beta 1, alpha 6 beta 1, alpha V beta 3. Attachment to laminin, a major adhesive protein in basement membranes, was studied in detail. Blocking monoclonal antibodies specific to different integrin receptor complexes showed that the alpha 6 beta 1 complex was important for MEC adhesion to laminin. In addition, blocking antibody also implicated the vitronectin receptor (alpha V beta 3) in laminin adhesion. We used ligand affinity chromatography of detergent-solubilized receptor complexes to further define receptor specificity. On laminin-Sepharose columns, we identified several integrin receptor complexes whose affinity for the ligand was dependent on the type of divalent cation present. Several beta 1 complexes, including alpha 1 beta 1, alpha 2 beta 1, and alpha 6 beta 1 bound strongly to laminin. In agreement with the antibody blocking experiments, alpha V beta 3 was found to bind well to laminin. However, unlike binding to its other ligands (e.g., vitronectin, fibrinogen, von Willebrand factor), alpha V beta 3 interaction with laminin did not appear to be Arg-Gly-Asp (RGD) sensitive. Finally, immunofluorescent staining demonstrated both beta 1 and beta 3 complexes in vinculin-positive focal adhesion plaques on the basal surface of MEC adhering to laminin-coated substrates. The results indicate that both these subfamilies of integrin heterodimers are involved in promoting MEC adhesion to laminin and the vascular basement membrane.     

Thymosin beta10 inhibits cell migration and capillary-like tube formation of human coronary artery endothelial cells

Thymosin beta10 is a cytoplasm G-actin sequestering protein whose functions are largely unknown. To determine the direct effects of exogenous thymosin beta10 on angiogenic potentials as endothelial cell migration and capillary-like tube formation, human coronary artery endothelial cells (HCAECs) were incubated with increasing doses of thymosin beta10 (25-100 ng/ml). By using a modified Boyden chamber assay, thymosin beta10 inhibited cell migration in a dose- and time-dependent manner with the maximal effect being a 36% reduction at 100 ng/ml as compared to controls (P < 0.01). In addition, thymosin beta10 (100 ng/ml) significantly inhibited the capillary-like tube-formation of HCAECs on Matrigel, showing a 21% reduction of the total tube length as compared to negative controls (P < 0.01). Furthermore, by using real time PCR analysis, thymosin beta10 significantly decreased mRNA levels of vascular endothelial growth factor (VEGF), VEGF receptor-1 (VEGFR-1) and integrin alphaV after 24 h treatment in HCAECs. By contrast, thymosin beta4 significantly increased HCAEC migration. These results indicate that thymosin beta10, but not thymosin beta4, have direct inhibitive effects on endothelial migration and tube formation that might be mediated via downregulation of VEGF, VEGFR-1 and integrin alphaV in HCAECs. This study suggests a potential therapeutic application of thymosin beta10 to the diseases with excessive angiogenesis such as cancer.     

The integrin complex alpha v beta 3 participates in the adhesion of microvascular endothelial cells to fibronectin.

Fibronectin is a major adhesive glycoprotein of the vascular basement membrane. Since fibronectin is also found in the interstitium, it may be important not only for attachment but also for endothelial cell migration during neovascularization. We have analyzed how human dermal microvascular endothelial cells use their diverse set of integrin receptors to interact with this ligand. Immunofluorescent staining with specific antibodies identified both beta 1 and beta 3 integrin receptor complexes in focal adhesion plaques on cells adhering to immobilized fibronectin. Adhesion assays with blocking monoclonal antibodies implicated both beta 1 and beta 3 complexes, specifically alpha 5 beta 1 and alpha v beta 3, in the initial adhesion of cells to fibronectin. Finally, ligand affinity chromatography of extracts of surface radiolabeled cells established that both alpha 5 beta 1 and alpha v beta 3 could bind to the 110-kDa cell-binding fragment of fibronectin. An additional receptor complex composed of an alpha v subunit and a beta 5-like subunit was also detected. These results provide evidence that microvascular endothelial cells use multiple integrin receptors, from several beta families, to attach to fibronectin surfaces.     

1-Deoxymannojirimycin inhibits capillary tube formation in vitro. Analysis of N-linked oligosaccharides in bovine capillary endothelial cells.

Capillary endothelial cells can be induced to form capillary-like structures in vitro by plating on fibronectin-coated dishes (Ingber, D. E., and Folkman, J. (1989) J. Cell Biol. 109, 317-330), thereby mimicking angiogenesis. To assess the role of glycoproteins bearing asparagine-linked oligosaccharides in this process, we tested the effect of oligosaccharide processing inhibitors on the formation of capillary tubes. Deoxymannojirimycin, a compound that prevents synthesis of hybrid and complex-type oligosaccharides, inhibited the formation of capillary tubes. In contrast, swainsonine, an inhibitor that blocks synthesis of complex- but not hybrid-type oligosaccharides, did not inhibit tube formation. Lectin affinity chromatography of 2-[3H] mannose-labeled glycopeptides from endothelial cells induced to form tubes did not reveal a striking difference in the spectrum of oligosaccharides compared to uninduced cells. Since endothelial cells formed tubes normally in the presence of swainsonine, we analyzed glycopeptides from swainsonine-treated induced and uninduced cells. Cells induced to form tubes were enriched in monosialylated hybrid-type oligosaccharides sensitive to alpha-fucosidase, beta-galactosidase, and beta-N-acetylhexosaminidase, suggestive of sialyl Lewis-X determinants. We used an enzyme-linked immunoassay to measure sialyl Lewis-X epitopes on capillary endothelial cells and found that both induced and uninduced cells expressed sialyl Lewis-X epitopes. Deoxymannojirimycin and, to a lesser extent, swainsonine reduced the level of sialyl Lewis-X epitopes in cells induced to form capillary tubes, but neither compound affected the level of epitopes in cell monolayers. We conclude that synthesis of at least hybrid-type oligosaccharides is required for capillary tube formation in vitro and that an increase in monosialylated, fucosylated glycans on asparagine-linked oligosaccharides occurs during this process.     

Cerebral microvascular endothelial cell tube formation: role of astrocytic epoxyeicosatrienoic acid release

Cerebral microvascular endothelial cells (CMVEC) form tubes when cocultured with astrocytes (AS). Therefore, it appears that AS may be important in mediating angiogenesis in the brain. We hypothesized that AS modulate CMVEC tube formation by releasing a soluble factor. Thymidine incorporation in cultured CMVEC increased 305% when incubated with 50% conditioned AS medium for 24 h [control: 52,755 +/- 4,838 counts per minute (cpm) per well, conditioned 161,082 +/- 12,099 cpm/well, n = 8]. Because our laboratory has previously shown that AS can produce epoxyeicosatrienoic acids (EETs), which are known mitogens, we investigated whether release of EETs by AS is responsible for tube formation in the CMVEC-AS coculture. AS were seeded on Lab-Tek slides, CMVEC were seeded on the AS the next day, and cultures were allowed to progress for another 5 days with and without cytochrome P-450 epoxygenase blockade by 17-octadecynoic acid (17-ODYA). Tube formation in cocultures receiving 17-ODYA was significantly inhibited compared with control (93.8%). These data suggest that tube formation requires the release of EETs by AS.     

Expression of integrin subunit beta1B in integrin beta1-deficient GD25 cells does not interfere with alphaVbeta3 functions

We have expressed the beta1B integrin subunit in beta1-deficient GD25 cells to examine beta1B functions without the interference of endogenous beta1A expression. As previously reported [Retta et al., 1998, Mol. Biol. Cell 9, 715-731], the beta1B integrins did not mediate cell adhesion under normal culture conditions, while the presence of 0.3 mM Mn(2+) allowed beta1B integrins to support adhesion. Mn(2+), as well as the small soluble peptide GRGDS, induced a beta1B conformation, which was recognized by the mAb 9EG7, a marker for active or ligand-bound integrins. beta1B integrins were found to localize to a subset of focal contacts in a ligand-independent manner on fibronectin, but not on vitronectin. However, clustering of beta1B did not induce tyrosine phosphorylation of FAK, p130(Cas), or paxillin, as studied by beta1B-mediated adhesion, to fibronectin in the presence of Mn(2+) or to anti-beta1 antibody in DMEM. Induction of ligand-occupied conformation by the GRGDS peptide during the adhesion to anti-beta1 antibody also failed to trigger FAK phosphorylation. Stimulation of tyrosine phosphorylation on FAK, p130(Cas), and paxillin by adhesion via integrin alphaVbeta3 to fibronectin or vitronectin was not disturbed in GD25-beta1B cells compared to the untransfected GD25 cells, nor were any negative effects of beta1B observed on alphaVbeta3-mediated cell attachment, spreading, and actin organization, or on the cell proliferation rate. These results show that the reported negative effects of beta1B on adhesive events do not apply to alphaVbeta3-dependent interactions and suggest that they may specifically act on beta1 integrins.     

Expression of chicken integrin beta 1 subunit in rat PC12 cells

We transfected rat pheochromocytoma (PC12) cells with a cDNA encoding chicken integrin beta 1 subunit. The chicken integrin beta 1 subunit produced in stable transfectants associated with two major alpha subunits of rat integrins to form interspecific chimeric receptors. These receptors mediated cell spreading and initial neurite outgrowth on laminin as did corresponding endogenous integrins, although they were slightly less effective in inducing cell adhesion to laminin. These results indicate that chicken integrin beta 1 may functionally substitute for beta 1 subunit of rat integrins in PC12 cells. Apparently, the structure of the integrin beta 1 subunit is highly conserved in the evolution of these species.     

Tyrosine phosphorylation of the integrin beta 3 subunit regulates beta 3 cleavage by calpain

Outside-in signaling of beta(3) integrins induces and requires phosphorylation at tyrosine 747 (Tyr(747)) and tyrosine 759 (Tyr(759)) of the beta(3) subunit, but the mechanism for this requirement is unclear. On the other hand, a key consequence of integrin signaling, cell spreading, is inhibited by calpain cleavage of beta(3) cytoplasmic domain. Here we show that beta(3) tyrosine phosphorylation inhibits calpain cleavage. Mutating both tyrosines to phenylalanine sensitizes beta(3) to calpain cleavage. Furthermore, phosphorylation at Tyr(747) and Tyr(759) of beta(3) in the focal adhesion sites and the leading edge of spreading platelets was differentially regulated. Selective dephosphorylation of Tyr(759) is associated with calpain cleavage at Tyr(759). Thus, one mechanism by which tyrosine phosphorylation promotes integrin signaling and cell spreading is its inhibition of calpain cleavage of the beta(3) cytoplasmic domain.     

Cell contact-dependent activation of alpha3beta1 integrin modulates endothelial cell responses to thrombospondin-1

Thrombospondin-1 (TSP1) can inhibit angiogenesis by interacting with endothelial cell CD36 or proteoglycan receptors. We have now identified alpha3beta1 integrin as an additional receptor for TSP1 that modulates angiogenesis and the in vitro behavior of endothelial cells. Recognition of TSP1 and an alpha3beta1 integrin-binding peptide from TSP1 by normal endothelial cells is induced after loss of cell-cell contact or ligation of CD98. Although confluent endothelial cells do not spread on a TSP1 substrate, alpha3beta1 integrin mediates efficient spreading on TSP1 substrates of endothelial cells deprived of cell-cell contact or vascular endothelial cadherin signaling. Activation of this integrin is independent of proliferation, but ligation of the alpha3beta1 integrin modulates endothelial cell proliferation. In solution, both intact TSP1 and the alpha3beta1 integrin-binding peptide from TSP1 inhibit proliferation of sparse endothelial cell cultures independent of their CD36 expression. However, TSP1 or the same peptide immobilized on the substratum promotes their proliferation. The TSP1 peptide, when added in solution, specifically inhibits endothelial cell migration and inhibits angiogenesis in the chick chorioallantoic membrane, whereas a fragment of TSP1 containing this sequence stimulates angiogenesis. Therefore, recognition of immobilized TSP1 by alpha3beta1 integrin may stimulate endothelial cell proliferation and angiogenesis. Peptides that inhibit this interaction are a novel class of angiogenesis inhibitors.     

Ascorbate inhibits NADPH oxidase subunit p47phox expression in microvascular endothelial cells

The production of reactive oxygen species (ROS) is central to the etiology of endothelial dysfunction in sepsis. Endothelial cells respond to infection by activating NADPH oxidases that are sources of intracellular ROS and potential targets for therapeutic administration of antioxidants. Ascorbate is an antioxidant that accumulates in these cells and improves capillary blood flow, vascular reactivity, arterial blood pressure, and survival in experimental sepsis. Therefore, the present study tested the hypothesis that ascorbate regulates NADPH oxidases in microvascular endothelial cells exposed to septic insult. We observed that incubation with Escherichia coli lipopolysaccharide (LPS) and interferon-gamma (IFNgamma) increased NADPH oxidase activity and expression of the enzyme subunit p47phox in mouse microvascular endothelial cells of skeletal muscle origin. Pretreatment of the cells with ascorbate prevented these increases. Polyethylene glycol-conjugated catalase and selective inhibitors of Jak2 also abrogated induction of p47phox. Exogenous hydrogen peroxide induced p47phox expression that was prevented by pretreatment of the cells with ascorbate. LPS+IFNgamma or hydrogen peroxide activated the Jak2/Stat1/IRF1 pathway and this effect was also inhibited by ascorbate. In conclusion, ascorbate blocks the stimulation by septic insult of redox-sensitive Jak2/Stat1/IRF1 signaling, p47phox expression, and NADPH oxidase activity in microvascular endothelial cells. Because endothelial NADPH oxidases produce ROS that can cause endothelial dysfunction, their inhibition by ascorbate may represent a new strategy for sepsis therapy.     

Distinct functions of integrin alpha and beta subunit cytoplasmic domains in cell spreading and formation of focal adhesions

Integrin-mediated cell adhesion often results in cell spreading and the formation of focal adhesions. We exploited the capacity of recombinant human alpha IIb beta 3 integrin to endow heterologous cells with the ability to adhere and spread on fibrinogen to study the role of integrin cytoplasmic domains in initiation of cell spreading and focal adhesions. The same constructs were also used to analyze the role of the cytoplasmic domains in maintenance of the fidelity of the integrin repertoire at focal adhesions. Truncation mutants of the cytoplasmic domain of alpha IIb did not interfere with the ability of alpha IIb beta 3 to initiate cell spreading and form focal adhesions. Nevertheless, deletion of the alpha IIb cytoplasmic domain allowed indiscriminate recruitment of alpha IIb beta 3 to focal adhesions formed by other integrins. Truncation of the beta 3 subunit cytoplasmic domain abolished cell spreading mediated by alpha IIb beta 3 and also abrogated recruitment of alpha IIb beta 3 to focal adhesions. This truncation also dramatically impaired the ability of alpha IIb beta 3 to mediate the contraction of fibrin gels. In contrast, the beta 3 subunit cytoplasmic truncation did not reduce the fibrinogen binding affinity of alpha IIb beta 3. Thus, the integrin beta 3 subunit cytoplasmic domain is necessary and sufficient for initiation of cell spreading and focal adhesion formation. Further, the beta 3 cytoplasmic domain is required for the transmission of intracellular contractile forces to fibrin gels. The alpha subunit cytoplasmic domain maintains the fidelity of recruitment of the integrins to focal adhesions and thus regulates their repertoire of integrins.     

Granzyme B cleavage of fibronectin disrupts endothelial cell adhesion,migration and capillary tube formation

Dysregulated angiogenesis contributes to the pathogenesis of chronic inflammatory diseases. Modulation of the extracellular matrix by immune-derived proteases can alter endothelial cell–matrix interactions as well as endothelial cell sprouting, migration and capillary formation. Granzyme B is a serine protease that is expressed by a variety of immune cells, and accumulates in the extracellular milieu in many chronic inflammatory disorders that are associated with dysregulated angiogenesis. Although granzyme B is known to cleave fibronectin, an essential glycoprotein in vascular morphogenesis, the role of granzyme B in modulating angiogenesis is unknown. In the present study, granzyme B cleaved both plasma fibronectin and cell-derived fibronectin, resulting in the release of multiple fibronectin fragments. Granzyme B cleavage of fibronectin resulted in a dose-dependent reduction in endothelial cell adhesion to fibronectin as well as reduced endothelial cell migration and tubular formation. These events were prevented when granzyme B activity was inhibited by a small molecule inhibitor. In summary, granzyme B-mediated cleavage of fibronectin contributes to attenuated angiogenesis through the disruption of endothelial cell — fibronectin interaction resulting in impaired endothelial cell migration and tubular formation.     

Vascular endothelial cell senescence mediated by integrin beta4 in vitro

To understand whether integrin beta4 is involved in vascular endothelial cell (VEC) senescence, we examined integrin beta4 level changes, as well as P53 and reactive oxygen species (ROS) levels and alterations of phosphatidylcholine-specific phospholipase C (PC-PLC) activity before and after knocking-down integrin beta4 by small interfering RNA. We found integrin beta4, P53 and ROS levels increased significantly, while Ca(2+)-independent PC-PLC activity obviously decreased during VEC senescence. On the other hand, integrin beta4 down-regulation attenuated the senescence phenotype and reversed Ca(2+)-independent PC-PLC activity, and P53 and ROS levels. The data suggested that integrin beta4 might mediate VEC senescence through depressing Ca(2+)-independent PC-PLC and elevating the levels of P53 and ROS.     

Caspase proteolysis of the integrin beta4 subunit disrupts hemidesmosome assembly, promotes apoptosis, and inhibits cell migration

Caspases are a conserved family of cell death proteases that cleave intracellular substrates at Asp residues to modify their function and promote apoptosis. In this report we identify the integrin beta4 subunit as a novel caspase substrate using an expression cloning strategy. Together with its alpha6 partner, alpha6beta4 integrin anchors epithelial cells to the basement membrane at specialized adhesive structures known as hemidesmosomes and plays a critical role in diverse epithelial cell functions including cell survival and migration. We show that integrin beta4 is cleaved by caspase-3 and -7 at a conserved Asp residue (Asp(1109)) in vitro and in epithelial cells undergoing apoptosis, resulting in the removal of most of its cytoplasmic tail. Caspase cleavage of integrin beta4 produces two products, 1) a carboxyl-terminal product that is unstable and rapidly degraded by the proteasome and 2) an amino-terminal cleavage product (amino acids 1-1109) that is unable to assemble into mature hemidesmosomes. We also demonstrate that caspase cleavage of integrin beta4 sensitizes epithelial cells to apoptosis and inhibits cell migration. Taken together, we have identified a previously unrecognized proteolytic truncation of integrin beta4 generated by caspases that disrupts key structural and functional properties of epithelial cells and promotes apoptosis.