The molecular balance between receptor tyrosine kinases Tie1 and Tie2 is dynamically controlled by VEGF and TNFα and regulates angiopoietin signalling.

Angiopoietin-1 (Ang1) signals via the receptor tyrosine kinase Tie2 which exists in complex with the related protein Tie1 at the endothelial cell surface. Tie1 undergoes regulated ectodomain cleavage in response to phorbol esters, vascular endothelial growth factor (VEGF) and tumour necrosis factor-α (TNFα). Recently phorbol esters and VEGF were found also to stimulate ectodomain cleavage of Tie2. Here we investigate for the first time the effects of factors activating ectodomain cleavage on both Tie1 and Tie2 within the same population of cells, and their impact on angiopoietin signalling. We find that phorbol ester and VEGF activated Tie1 cleavage within minutes followed by restoration to control levels by 24 h. However, several hours of PMA and VEGF treatment were needed to elicit a detectable decrease in cellular Tie2, with complete loss seen at 24 h of PMA treatment. TNFα stimulated Tie1 cleavage, and induced a sustained decrease in cellular Tie1 over 24 h whilst increasing cellular Tie2. These differential effects of agonists on Tie1 and Tie2 result in dynamic modulation of the cellular Tie2∶Tie1 ratio. To assess the impact of this on Ang1 signalling cells were stimulated with VEGF and TNFα for differing times and Ang1-induced Tie2 phosphorylation examined. Elevated Tie2∶Tie1, in response to acute VEGF treatment or chronic TNFα, was associated with increased Ang1-activated Tie2 in cells. These data demonstrate cellular levels of Tie1 and Tie2 are differentially regulated by pathophysiologically relevant agonists resulting in dynamic control of the cellular Tie2∶Tie1 balance and modulation of Ang1 signalling. These findings highlight the importance of regulation of signalling at the level of the receptor. Such control may be an important adaptation to allow modulation of cellular signalling responses in systems in which the activating ligand is normally present in excess or where the ligand provides a constitutive maintenance signal.

Studies on the morphology and spreading of human endothelial cells define key inter- and intramolecular interactions for talin1.

Talin binds to and activates integrins and is thought to couple them to cytoskeletal actin. However, functional studies on talin have been restricted by the fact that most cells express two talin isoforms. Here we show that human umbilical vein endothelial cells (HUVEC) express only talin1, and that talin1 knockdown inhibited focal adhesion (FA) assembly preventing the cells from maintaining a spread morphology, a phenotype that was rescued by GFP-mouse talin1. Thus HUVEC offer an ideal model system in which to conduct talin structure/function studies. Talin contains an N-terminal FERM domain (comprised of F1, F2 and F3 domains) and a C-terminal flexible rod. The F3 FERM domain binds beta-integrin tails, and mutations in F3 that inhibited integrin binding (W359A) or activation (L325R) severely compromised the ability of GFP-talin1 to rescue the talin1 knockdown phenotype despite the presence of a second integrin-binding site in the talin rod. The talin rod contains several actin-binding sites (ABS), and mutations in the C-terminal ABS that reduced actin-binding impaired talin1 function, whereas those that increased binding resulted in more stable FAs. The results show that both the N-terminal integrin and C-terminal actin-binding functions of talin are essential to cell spreading and FA assembly. Finally, mutations that relieve talin auto-inhibition resulted in the rapid and excessive production of FA, highlighting the importance of talin regulation within the cell.

Effects of angiopoietins-1 and -2 on the receptor tyrosine kinase Tie2 are differentially regulated at the endothelial cell surface.

Angiopoietin-1 (Ang1) and Ang2 are ligands for the receptor tyrosine kinase Tie2. Structural data suggest that the two ligands bind Tie2 similarly. However, in endothelial cells Ang1 activates Tie2 whereas Ang2 can act as an apparent antagonist. In addition, each ligand exhibits distinct kinetics of release following binding. These observations suggest that additional factors influence function and binding of angiopoietins with receptors in the cellular context. Previous work has shown that Ang1 binding and activation of Tie2 are inhibited by Tie1, a related receptor that complexes with Tie2 in cells. In this study we have investigated binding of Ang1 and Ang2 to Tie2 in endothelial cells. In contrast to Ang1, binding of Ang2 to Tie2 was found to be not affected by Tie1. Neither PMA-induced Tie1 ectodomain cleavage nor suppression of Tie1 expression by siRNA affected the ability of Ang2 to bind Tie2. Analysis of the level of Tie1 co-immunoprecipitating with angiopoietin-bound Tie2 demonstrated that Ang2 can bind Tie2 in Tie2:Tie1 complexes whereas Ang1 preferentially binds non-complexed Tie2. Stimulation of Tie1 ectodomain cleavage did not increase the agonist activity of Ang2 for Tie2. Similarly, the Tie2-agonist activity of Ang2 was not affected by siRNA suppression of Tie1 expression. Consistent with previous reports, loss of Tie1 ectodomain enhanced the agonist activity of Ang1 for Tie2. Importantly, Ang2 was still able to antagonize the elevated Ang1-activation of Tie2 that occurs on Tie1 ectodomain loss. Together these data demonstrate that Ang1 and Ang2 bind differently to Tie2 at the cell surface and this is controlled by Tie1. This differential regulation of angiopoietin binding allows control of Tie2 activation response to Ang1 without affecting Ang2 agonist activity and maintains the ability of Ang2 to antagonize even the enhanced Ang1 activation of Tie2 that occurs on loss of Tie1 ectodomain. This provides a mechanism by which signalling through Tie2 can be modified by stimuli in the cellular microenvironment.

Roles of the receptor tyrosine kinases Tie1 and Tie2 in mediating the effects of angiopoietin-1 on endothelial permeability and apoptosis.

Angiopoietin-1 (Ang1) has key roles in development and maintenance of the vascular system. The ligand is a potent inhibitor of vascular leakage and suppresses endothelial apoptosis and vessel regression. Ang1 was originally identified as a ligand for the receptor tyrosine kinase Tie2. Recently however Ang1 has also been found to activate the related tyrosine kinase Tie1. The contribution of Tie1 to mediating the effects of Ang1 on endothelial function is not known. In this study we used an siRNA approach to investigate the relative importance of Tie1 and Tie2 in transducing the effects of Ang1 on monolayer permeability and induction of apoptosis in human endothelial cells. siRNA directed against either Tie1 or Tie2 suppressed expression of each respective receptor by more than 90%. Ang1 inhibited endothelial monolayer permeability and this effect was prevented by suppression of Tie2 expression. In contrast, Ang1 inhibition of permeability was not affected by suppression of Tie1 expression. The ability of Ang1 to inhibit induction of apoptosis in response to serum deprivation was completely blocked by suppression of Tie2 expression, but not diminished by suppression of Tie1 expression. Taken together these data demonstrate that Tie2 mediates the inhibitory effects of Ang1 on endothelial permeability and apoptosis. The data also demonstrates that Tie1 does not transduce anti-apoptotic or anti-permeability effects of Ang1 in endothelial cells.

Vascular endothelial growth factor and angiopoietins in neurovascular regeneration and protection following stroke.

Vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang1) were originally identified as endothelial-specific ligands regulating key functions of the vasculature important in stroke. There is increasing evidence that these ligands also exert effects on neurons. Here we review the neuronal effects of VEGF and Ang1 and highlight their potential for therapeutic manipulation in stroke. VEGF stimulates angiogenesis whereas Ang1 suppresses leakage, inflammation and regression of microvessels. Expression of both ligands change dramatically in the brain in experimental stroke, correlating with increased vascular leakage and inflammation. In addition to vascular effects, VEGF can stimulate survival, migration and proliferation of neurons suggesting roles in neural protection and possible therapeutic applications, an idea supported by preclinical studies. Recent reports now demonstrate that Ang1 can also act directly on neurons and enhance neural repair. The realization that VEGF and Ang1 have effects on both neural and vascular compartments impacted by stroke provides new opportunities for therapeutic manipulation to promote neuroprotection and extend the thrombolytic window, as well as stimulating neurogenesis and revascularization.

AIF: a multifunctional cog in the life and death machine.

Mitochondria have a dual role in cellular life and death as life-promoting energy providers and as contributors to programmed cell death (apoptosis). The precise sequence of events resulting in the permeabilization of the mitochondrial membrane and the release of mitochondrial resident proteins remains an actively explored topic. Hansen and Nagley describe results from mammalian cells and from the nematode C. elegans that lead to a feedforward model for mitochondrial destabilization. Furthermore, they describe the mitochondrial and apoptotic functions of several proteins released from mitochondria during progression toward cell death.

On the release of cytochrome c from mitochondria during cell death signaling.

Mitochondria play key roles in apoptosis, a central step being the release of cytochrome c (cyt c) across the outer mitochondrial membrane into the cytoplasm. We review this process in terms of the influences that induce mitochondria to release cyt c, the possible mechanisms of such release and the downstream consequences for caspase activation. The contributions of members of the Bcl-2 family in regulating mitochondrial activities relevant to apoptotic signaling are considered. Antiapoptotic members, such as Bcl-2 itself, are antagonistic to other family members, which prominently include Bax amongst a host of other proapoptotic proteins homologous to Bcl-2. Focus is placed on technical methods of determining cyt c release, which encompass cell fractionation, biochemistry, immunochemistry and confocal microscopy [including observations of release in real time using cyt c-green fluorescent protein (GFP) fusion proteins]. The advantages and potential pitfalls of the various approaches are discussed, with some emphasis on the use of cyt c-GFP fusions and the determination of the characteristics of the putative outer membrane pore through which cyt c and other mitochondrial proteins with proapoptotic functions may pass. The richness of this field relating to mitochondria and cell death is brought out by consideration of studies carried out in mammalian and yeast cells.