The angiopoietins (Ang-1 and Ang-2) have been identified as agonistic and antagonistic ligands of the endothelial receptor tyrosine kinase Tie2, respectively. Both ligands have been demonstrated to induce translocation of Tie2 to cell-cell junctions. However, only Ang-1 induces Tie2-dependent Akt activation and subsequent survival signaling and endothelial quiescence. Ang-2 interferes negatively with Ang-1/Tie2 signaling, thereby antagonizing the Ang-1/Tie2 axis. Here, we show that both Ang-1 and Ang-2 recruit beta3 integrins to Tie2. This co-localization is most prominent in cell-cell junctions. However, only Ang-2 stimulation resulted in complex formation among Tie2, alphavbeta3 integrin, and focal adhesion kinase as evidenced by co-immunoprecipitation experiments. Focal adhesion kinase was phosphorylated in the FAT domain at Ser(910) upon Ang-2 stimulation and the adaptor proteins p130Cas and talin dissociated from alphavbeta3 integrin. The alphavbeta3 integrin was internalized, ubiquitinylated, and gated toward lysosomes. Taken together, the experiments define Tie2/alphavbeta3 integrin association-induced integrin internalization and degradation as mechanistic consequences of endothelial Ang-2 stimulation.
"We have previously demonstrated that Ang-2 stimulation of endothelial cell/smooth muscle cell co-culture spheroids results in destabilization and subsequent loss of association between the two cell types with loosened inter-endothelial junctions, which do not involve VE-cadherin but Tie-2-integrin complexes , . Ang-1* stimulation has also been shown to reduce inter-endothelial gaps  and Ang-2 transgenic mice resemble the Tie-2/Ang-1 knockout mice demonstrating a qualitatively similar phenotype , , . "
[Show abstract][Hide abstract] ABSTRACT: Genetic experiments (loss-of-function and gain-of-function) have established the role of Angiopoietin/Tie ligand/receptor tyrosine kinase system as a regulator of vessel maturation and quiescence. Angiopoietin-2 (Ang-2) acts on Tie2-expressing resting endothelial cells as an antagonistic ligand to negatively interfere with the vessel stabilizing effects of constitutive Ang-1/Tie-2 signaling. Ang-2 thereby controls the vascular response to inflammation-inducing as well as angiogenesis-inducing cytokines. This study was aimed at assessing the role of Ang-2 as an autocrine (i.e. endothelial-derived) regulator of rapid vascular responses (within minutes) caused by permeability-inducing agents. Employing two independent in vivo assays to quantitatively assess vascular leakage (tracheal microsphere assay, 1-5 min and Miles assay, 20 min), the immediate vascular response to histamine, bradykinin and VEGF was analyzed in Ang-2-deficient (Ang-2(-/-)) mice. In comparison to the wild type control mice, the Ang2(-/-) mice demonstrated a significantly attenuated response. The Ang-2(-/-) phenotype was rescued by systemic administration (paracrine) of an adenovirus encoding Ang-2. Furthermore, cytokine-induced intracellular calcium influx was impaired in Ang-2(-/-) endothelioma cells, consistent with reduced phospholipase activation in vivo. Additionally, recombinant human Ang-2 (rhAng-2) alone was unable to induce vascular leakage. In summary, we report here in a definite genetic setting that Ang-2 is critical for multiple vascular permeability-inducing cytokines.
PLoS ONE 08/2013; 8(8):e70459. DOI:10.1371/journal.pone.0070459 · 3.23 Impact Factor
"with subsequent integrin internalization and degradation . We and others have shown that Ang-2 levels in plasma from critically-ill septic patients correlate with the extent of pulmonary vascular leak and acute lung injury  , increase with the severity of multiple-organ dysfunction syndrome  , and independently predict mortality in the intensive care unit (ICU)      . "
[Show abstract][Hide abstract] ABSTRACT: Introduction:
Endothelial activation leading to vascular barrier dysfunction and organ failure is a well-recognized complication of cardiovascular surgery with cardiopulmonary bypass (CPB). The endothelial-specific angiopoietin-Tie2 ligand-receptor system has been identified as a non-redundant regulator of endothelial activation. Binding of angiopoietin-2 (Ang-2) to the Tie2 receptor antagonizes Tie2 signaling and renders the endothelial barrier responsive to pro-inflammatory cytokines. We aimed to study the time course and potential triggering factors of Ang-2 release after CPB, as well as the association of Ang-2 changes with surrogates of increased vascular permeability, organ dysfunction, and outcome.
Serum levels of Ang-2 from 25 adult patients (140 screened) were measured before and at 0, 12, and 24h following CPB procedure by in-house immuno-luminometric assay (ILMA), and compared with indices of organ dysfunction, duration of mechanical ventilation (MV), length of stay (LOS) in the intensive care unit (ICU), and hospital mortality. The effect of Ang-2 was studied in vitro by incubating high Ang-2 patient serum with endothelial cells (EC).
Ang-2 levels steadily increased from 2.6 ± 2.4 ng/mL at 0 h up to 7.3 ± 4.6 ng/mL at 24h following CPB (P<0.001). The release of Ang-2 correlated with the duration of CPB, aortic cross-clamp time, and post-CPB lactate levels. Changes in Ang-2 during follow-up correlated with partial pressure of oxygen in arterial blood (PaO(2))/fraction of inspired oxygen (FiO(2)) ratio, alveolar-arterial oxygen tension difference (AaDO(2)), hemodynamics, fluid balance, and disease severity measures. Ang-2 levels at 12h predicted the duration of MV, ICU-LOS, and hospital mortality. High Ang-2 patient sera disrupted EC architecture in vitro, an effect reversed by treatment with the competitive Tie2 ligand angiopoietin-1 (Ang-1).
Collectively, our results suggest that Ang-2 is a putative mediator of endothelial barrier dysfunction after CPB. These findings suggest that targeting the Ang/Tie2 pathway may mitigate organ dysfunction and improve outcome in patients undergoing CPB.
"However, it was not determined if α v β 3 integrin was ubiquitylated at the cell surface, resulting in receptor internalization or if ubiquitylation occurred post internalization. Additionally, α v β 3 integrin colocalized with the lysosome marker LAMP1 after 1 hour of angiopoietin-2 treatment, but whether α v β 3 integrin trafficking to lysosomes required ubiquitylation was not determined (Thomas et al., 2010). "
[Show abstract][Hide abstract] ABSTRACT: Adenovirus relies on numerous interactions between viral and host cell proteins to efficiently enter cells. Undoubtedly, post-translational modifications of host and cellular proteins can impact the efficiency of this cell entry process. Ubiquitylation, once simply thought of as a modification targeting proteins for proteasomal degradation, is now known to regulate protein trafficking within cells, protein-protein interactions and cell signalling pathways. Accumulating evidence suggests that protein ubiquitylation can influence all stages of the life cycle of other viruses such as cell entry, replication and egress. Until recently, the influence of ubiquitylation has only been documented during adenovirus replication. This review highlights the most recent evidence demonstrating direct engagement of host ubiquitylation and SUMOylation machinery by adenovirus during cell entry. Additionally, potential roles for host protein ubiquitylation and the potential for adenovirus regulation of host ubiquitylation machinery during cell entry are discussed.
Biology of the Cell 03/2012; 104(3):188-98. DOI:10.1111/boc.201100096 · 3.51 Impact Factor
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