Angiopoietin/Tie Proteins

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Angiopoietin/Tie Proteins

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Angiopoietin/Tie Proteins Background

Endothelial cells are essential to angiogenesis and their functions are regulated, in part, by growth factor stimulation of the cell surface receptor tyrosine kinases (RTKs). The Tie (tyrosine kinase with immunoglobulin and epidermal growth factor homology domains) family represents a distinct class of type I transmembrane RTKs expressed primarily by endothelial cells. There are two identified members, Tie-1 and Tie-2. Four secreted growth factors, termed angiopoietins, bind exclusively to Tie-2. Tie-1 is considered an orphan receptor, however it has been reported to display affinity to some members of the angiopoietin family.
Structurally, Tie-1 and Tie-2 are similar with molecular weights of 135 and 150 kDa, respectively. They are 76% identical in their extracellular region, which consists of three immunoglobulin (Ig)–like domains, separated by three tandem epidermal growth factor (EGF)-like cysteine repeats, and followed by three fibronectin type III homology domains. Selective deletion studies indicate that the Ig-like and EGF-like repeats are responsible for ligand recognition and binding. The cytoplasmic tails of Tie-1 and Tie-2 are 33% identical and consist of a split tyrosine kinase domain that is responsible for activating the downstream signaling cascades after autophosphorylation.

Four ligands have been identified for the Tie RTKs: Angiopoietin (Ang) -1, Ang-2 and Ang-4 in humans and Ang-3 in mice. Ang-1 and Ang-2 share 60% homology and are structurally similar, consisting of three distinct regions: a coiled-coil domain at the NH2-terminus, a short linker peptide, and a COOH-terminal domain homologous to the COOH-terminal domain of fibrinogen. The coiled-coil domain is responsible for the homo-oligomerization of the angiopoietins and the fibrinogen homology domain (FHD) mediates Tie-2 binding. Despite binding to the same site on Tie-2 with similar affinity, the second Ig-like domain, Ang-1 and Ang-2 induce different biological responses, which first became evident by the distinct phenotypes of Ang-1 and Ang-2 null mice. Targeted disruption of Ang-1 results in a phenotype slightly less severe than, but similar to, the disruption of Tie-2, indicating a role for Ang-1 as an agonist for Tie-2. Ultrastructural analysis of these mutants revealed that endothelial cells are weakly associated with the underlying matrix and peri-endothelial supporting cells, suggesting that Ang-1 regulates endothelial-matrix interactions and vessel stability. Interestingly, Ang-2 is dispensable to vascular development and over-expression of the gene results in vascular defects similar to the disruption of Ang-1 or Tie-2, indicating an antagonistic role of Ang-2 on Tie-2 activity during embryogenesis. Thus, Ang-2 represents the first naturally occurring antagonist of RTKs and highlights the importance of tightly regulating Tie-2 signaling during normal development. 
In situ hybridization of developing embryos revealed that Ang-1 expression is concentrated primarily in the heart early in development and more widespread in adulthood. Ang-2 expression, on the other hand, is localized to areas of frequent vascular remodeling including the ovary, placenta, and uterus. The sources of Ang-1 and Ang-2 also differ; smooth muscle cells and endothelial cells secrete Ang-1 and Ang-2, respectively. Upon secretion, Ang-1 is incorporated into the ECM, where it is believed to serve as a localized agonist of Tie-2. Ang-2, on the other hand, is stored in Weibel-Palade bodies located in the cytoplasm of endothelial cells and is quickly released upon stimulation.

The angiopoietins can mediate wide-ranging effects on endothelial cell biology and, as demonstrated in later chapters of this thesis, on the activity of tumor cells. As mentioned briefly above, only binding of Ang-1 to Tie-2 leads to definitive receptor activation and downstream signaling. Ang-2, however, can affect angiogenesis by inhibiting Tie-2 activation and functioning in concert with localized VEGF to promote EC proliferation. Ultimately, signals through the Tie-2 receptor lead to alterations in endothelial cell survival, migration, quiescence and permeability.
Ang-1 has been shown to inhibit apoptosis and enhance survival of endothelial cells in a Tie- 2 dependent manner. More specifically, Ang-1-induced tyrosine phosphorylation of Tie-2 stimulates the recruitment of a number of adaptor proteins, including growth factor receptor-bound protein 2 (GRB2) and the p85 subunit of the phosphoinositide 3-kinase (PI3K), ultimately leading to the phosphorylation of Ser473 on AKT. Subsequently, AKT promotes EC survival through the induction of the endothelial nitric oxide synthase (eNOS), up-regulation of survivin, and the suppression of pro-apoptotic factors, such as caspase-9 and BAD. In addition, AKT activation leads to the inhibitory phosphorylation of forkhead transcription factor FOXO1, which is a known Ang-2 transcription factor. Thus, Ang-1 promotes a negative feedback loop on production of Ang-2 by endothelial cells, which enhances endothelial survival and maintenance. Conversely, PI3K inhibition leads to an Ang-2- mediated positive feedback loop in which FOXO1 activation induces transcription of Ang-2 to promote vessel destabilization and remodeling.
Studies have demonstrated that Ang-1 is chemotactic for endothelial cells and can induce EC migration through two Tie-2 dependent mechanisms. First, similar to the induction of endothelial survival, Tie-2 can stimulate cell motility through the activation of PI3K and subsequent downstream effectors. Second, Tie-2 phosphorylation can result in the tyrosine phosphorylation of Dok-R, leading to the formation of a Dok-R-Nck-p21-activating kinase (Pak/Pak1)/Tie-2 complex. In the absence of cell contacts (i.e. during vessel sprouting), Ang-1 mediated Tie-2 activation induces the relocation of the complex to cell-matrix contacts and induces Erk phosphorylation, resulting in proliferation and migration of endothelial cells. Interestingly, both Ang-1 and Ang-2 are chemotactic for fibroblasts ectopically expressing Tie-2.
The mechanism underlying Ang-1 mediated recruitment of peri-endothelial cells is not well elucidated. Heparin-binding epidermal-like growth factor (HB-EGF), hepatocyte growth factor (HGF), and serotonin have been shown to play roles in recruitment of smooth muscle cells to neovessels. In addition, in the presence of cell-cell contacts, a situation that mimics the quiescent vasculature, Ang-1 induces Tie-2 phosphorylation and trans-association of the Ang-1/Tie-2 complex with Tie-2 expressed on neighboring cells. Subsequent activation of the AKT-FOXO1 pathway induces EC survival and promotes blood vessel stability.
Ang-1 mediated signaling is also responsible for the changes in endothelial cell permeability. Ang-1 induced activation of Tie-2 enhances the recruitment of peri-endothelial cells to vessels, as well as strengthens the adherens junctions and tight junctions in the resting endothelial layer, thereby decreasing vessel leakiness. In contrast, Ang-2 mediated Tie-2 inhibition results in pericyte dropout and increased vessel permeability.

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