Angiopoietins and Angiopoietin-like Protein Families Background
Angiopoietins are the ligands of Tie-2 receptors and comprise four members, angiopoietin-1, -2, -3 and -4. The structure of these proteins consists of a fibrinogen (FN)-like domain responsible for Tie-2 receptor binding that contains the most conserved sequence between all angiopoietin members, a central coiled-coil domain involved in oligomerizing these FN-like domains through disulfide linkages and a short hydrophobic amino terminal domain having both secretory and superclustering functions, which thus generates variable order multimers in the interstitial space. Ang-1 was the first family member to be identified in 1996, followed by Ang-2 one year later. Although Ang-l-induced Tie-2 activation is mainly mediated by full length Ang-1 (1.5 Kb), which spans 498 amino acids (aas), three isoforms generated from alternative splicing have recently been cloned and designated as Ang-1-0.7 Kb (154 aas), Ang-1-0.9 Kb (285 aas) and Ang-1-1.3 Kb (367 aas). The roles of these isoforms are not well characterized, but recent evidence indicates that the 0.9 and 1.3 Kb isoforms, which partially lack the coiled-coiled and FN-like domains respectively, function as dominant negative regulators of full length Ang-1. Similarly, a splice variant for Ang-2, which partially lacks the coiled-coil domain and acts as a dominant negative regulator of Ang-1 has been recently characterized and designated as Ang- 2443 in humans and as Ang-2B in chickens.
Biological functions of angiopoietins during embryonic development
Targeted disruption of the Angptl gene produces similar phenotypic manifestations as those seen in TIE-2-/- mice, including gross vascular abnormalities and embryonic lethality at E l2.5. Overexpression of Ang-1 in the skin of transgenic mice corroborate the previous finding, as these mice exhibit increased vascularity and reduction of vascular leakage, indicating that Ang-1 promotes branching and maturation of blood vessels and maintains vascular integrity. In addition, Ang-1-/- mice embryos are characterized by rounded ECs and the absence of ECM and periendothelial cells, demonstrating that Ang-1 promotes vascular integrity in part by recruiting extraendothelial support. In culture, Ang-1 promotes EC migration, differentiation on collagen or fibrin matrices and sprouting angiogenesis and, under certain circumstances, is mitogenic for ECs. Ang-1-mediated EC adhesion and migration can also be mediated through integrin activation independently of Tie-2. Recently Ang-1, along with the orphan receptor Tie-1, was reported to establish polarity during angiogenesis, as Ang-l-/- Tie-l-/- mice had disorganized vessels only in the righthand side venous system at E8.5-E9.5.
Contradictory results have thus far been obtained regarding the biological functions of Ang-2. Initial reports suggested that overexpression of Ang-2 in mice elicits phenotypical changes similar to those found in Tie-2-/- and Ang-1-/- mice, involving poor maturation of the vasculature, EC apoptosis and embryonic lethality at E9.5-E10.5. However, Ang-2 knockout mice develop chylous ascites shortly after birth with major defects in lymphogenesis, angiogenic and inflammation. More recently, Ang-2 knockout pups have been shown to develop dysmorphic cortical peritubular capillaries in the kidney. These results demonstrate a main role for Ang-2 during late embryogenesis as well as during postnatal angiogenesis and lymphogenesis. In cultured ECs, although initial studies reported a proapoptotic and antiangiogenic role for Ang-2, recent studies have shown that Ang-2 can induce migration of EC monolayers and promote robust migration and sprouting of ECs in three-dimensional cultures. These contrasting roles of Ang-2 might depend on the presence of other angiogenic cytokines, on ECM components and on the dosage of Ang-2. It is thus becoming increasingly clear that the biological roles of Ang-2 are context-dependent.
The physiological roles of Ang-3 and Ang-4 during development are currently unknown as there are no knockout or transgenic animals of either angiopoietins, although in-vitro studies suggest that they might have similar roles in mediating angiogenesis as those of Ang-2 and Ang-1, respectively.
Angiopoietins and Tie receptors
In adults, increased Tie-2 expression is observed in the leading edges of sprouting vessels under both physiological (e.g., wound healing and menses) and pathological (e.g., tumor) conditions, while in the quiescent vasculature, Tie-2 phosphorylation levels remain high due to constitutive secretion of Ang-1 by periendothelial cells. This shows that activation of Tie-2 is required to maintain both angiogenesis and vascular homeostasis in adults.
Constitutive Ang-1 expression also plays an important role in potentiating the angiogenic effects of VEGF, as was first demonstrated in a corneal neovascularization preparation. In adults, the roles of Ang-1 have been mainly demonstrated during pathological conditions, such as ischemia-induced angiogenesis, where injection of Ang-1 expression vector in rabbits enhanced collateral vessel formation. Ang-1 also exerts anti-inflammatory roles during rheumatoid arthritis, edema, cardiac allograft arteriosclerosis and endotoxic shock. This anti-inflammatory role of Ang-1 involves tightening of EC junctions through the mobilization of platelet/endothelial cell adhesion molecule-1 (PECAM-1) to junctional areas and inhibition of VE-cadherin (VE-cadherin) expression, as well as the inhibition of phosphorylation of these proteins. Furthermore, Ang-1 attenuates the expression of tumor necrosis factor alpha (TNFα)- and VEGF-induced adhesion molecules (ICAM-1, VCAM-1 and E selectin), reduces leukocyte adhesion to ECs, and lowers TNFα-induced tissue factor expression. In addition, various cancer cell lines and tumor tissues have recently been found to overexpress Ang-1, although the functional relevance is unclear.
Tie-1 has also been implicated in modulating adult vascular functions. Recent evidence suggests that both Tie-1 and Tie-2 collaborate to reendothelize the vasculature following balloon carotid injury in the rat and to promote postnatal bone marrow haematopoiesis (387;388). Whether these effects are modulated by Ang-1, however, are unknown.
Ang-2 appears to mainly have a role during postnatal angiogenesis, particularly at sites of vascular remodeling, since Ang-2 mRNA is expressed in the placenta, uterus and ovaries of adult mammals. In addition, persistent and elevated Ang-2 expression has been described in wound healing regions of adult animals. Moreover, numerous reports have confirmed that proangiogenic stimuli such as hypoxia significantly upregulate Ang-2 but not Ang-1 expression, and that Ang-2 plays an important role during kidney and retinal vascular maturation. Furthermore, Ang-2 induces the differentiation of ECs grown in fibrin matrices, indicating that Ang-2 may promote angiogenesis in cultured ECs. The biological effects of Ang-2 might depend on the presence or absence of potent angiogenic inducers, most notably VEGF, resulting in cell migration or apoptosis and subsequent vessel regression, respectively. Under pathological conditions, persistent and elevated Ang-2 expression has been described in various cancers. In inflammatory settings, Ang-2 is elevated during ischemia-induced retinal neovascularization and modulates TNFα-mediated survival, angiogenesis and leukocyte-EC interactions. Finally, it has been recently proposed that Ang-2 can directly activate EPCs and promote their “homing” to denuded sites, as occurs during myocardial infarctions.
In adults, the similar level of activation of Tie-2 receptors by Ang-3 and Ang-4 as compared with Ang-2 and Ang-1, respectively, implies similar functions. However, the roles of Ang-3 and Ang-4 during adult physiological and pathological angiogenesis are not as well characterized as for Ang-1 and Ang-2. Ang-3 has recently been shown to inhibit tumor angiogenesis, presumably by promoting tumor cell apoptosis and inhibiting EC proliferation and survival. These effects of Ang-3 are partly mediated by antagonizing Ang-1 and VEGF signaling. Another study demonstrated that Ang-4 levels are downregulated in a murine model of LPS-induced acute lung injury, where VEGF levels are simultaneously upregulated, suggesting that like Ang-1, Ang-4 attenuates VEGF-induced inflammation. It should be pointed out, however, that the authors used an Ang-4 antibody on mice tissue, indicating they were most likely detecting Ang-3.