Eph receptor tyrosine kinases and their ligands (ephrins) are key players during the development of the embryonic vasculature; however, their role and regulation in adult angiogenesis remain to be defined. Both receptors and ligands have been shown to be up-regulated in a variety of tumors. To address the hypothesis that hypoxia is an important regulator of Ephs/ephrins expression, we developed a mouse skin flap model of hypoxia. We demonstrate that our model truly represents segmental skin hypoxia by applying four independent methods: continuous measurement of partial cutaneous oxygen tension, monitoring of tissue lactate/pyruvate ratio, time course of hypoxia-inducible factor-1alpha (HIF-1alpha) induction, and localization of stabilized HIF-1alpha by immunofluorescence in the hypoxic skin flap. Our experiments indicate that hypoxia up-regulates not only HIF-1alpha and vascular endothelial growth factor (VEGF) expression, but also Ephs and ephrins of both A and B subclasses in the skin. In addition, we show that in Hep3B and PC-3 cells, the hypoxia-induced up-regulation of Ephs and ephrins is abrogated by small interfering RNA-mediated down-regulation of HIF-1alpha. These novel findings shed light on the role of this versatile receptor/ligand family in adult angiogenesis. Furthermore, our model offers considerable potential for analyzing distinct mechanisms of neovascularization in gene-targeted mice.
Vihanto et al., FASEB J, 2005
Eph receptor tyrosine kinases are key players during the development of the embryonic vasculature; however, their role and regulation in adult angiogenesis remain to be defined. Caveolae are flask-shaped invaginations of the cell membrane; their major structural protein, caveolin-1, has been shown to regulate signaling molecules localized in these micro-domains. The interaction of caveolin-1 with several of these proteins is mediated by the binding of its scaffolding domain to a region containing hydrophobic amino acids within these proteins. The presence of such a motif within the EphB1 kinase domain prompted us to investigate the caveolar localization and regulation of EphB1 by caveolin-1. We report that EphB1 receptors are localized in caveolae, and directly interact with caveolin-1 upon ligand stimulation. This interaction, as well as EphB1-mediated activation of extracellular-signal-regulated kinase (ERK), was abrogated by overexpression of a caveolin-1 mutant lacking a functional scaffolding domain. Interaction between Ephs and caveolin-1 is not restricted to the B-subclass of receptors, since we show that EphA2 also interacts with caveolin-1. Furthermore, we demonstrate that the caveolin-binding motif within the kinase domain of EphB1 is primordial for its correct membrane targeting. Taken together, our findings establish caveolin-1 as an important regulator of downstream signaling and membrane targeting of EphB1.
Vihanto et al., J Cell Science, 2006
Eph receptor tyrosine kinases and their ligands (ephrins) are involved in cancerogenesis and pathological neovascularization. However, the mechanisms underlying their role in tumor formation and metastasis remain to be defined. Here, we demonstrated that stimulation of EphB1 with ephrinB1/Fc led to a marked downregulation of EphB1 protein, a process blocked by the lysosomal inhibitor bafilomycin. Following ephrinB1 stimulation, the ubiquitin ligase Cbl was recruited by EphB1, then phosphorylated. Both Cbl phosphorylation and EphB1 ubiquitination were blocked by the Src inhibitor PP2. Overexpression of wild-type Cbl, but not of 70-Z mutant lacking ligase activity, enhanced EphB1 ubiquitination and degradation. This negative regulation required the tyrosine kinase activity of EphB1, as kinase dead EphB1-K652R was resistant to Cbl. GST binding experiments showed that Cbl bound to EphB1 through its TKB domain. In aggregate, we demonstrated that Cbl induces the ubiquitination and lysosomal degradation of activated EphB1, a process requiring EphB1 and Src kinase activity. To our knowledge, this is the first study dissecting the molecular mechanisms leading to EphB1 downregulation, thus paving the way to new means of modulating their angiogenic and tumorigenic properties.
Fasen et al., Traffic., 2007
PD Dr. Uyen Huynh-Do
Swiss National Science Foundation, Project no. 105871
The aim of our study was to investigate the phenomenon of intussusceptive angiogenesis with focus on its molecular regulation by VEGFR/PDGFRβ pathways and biological significance for glomerular recovery after acute injury. Glomerular healing by intussusception was examined in a particular setting of Thy1.1 nephritis, where the lysis of mesangial cells results in an initial collapse and successive rebuilding of glomerular capillary structure.
Restoration of capillary structure after induction of Thy1.1 nephritis occurred by intussusceptive angiogenesis. Intussusception resulted in (i) rapid expansion of the capillary plexus with reinstatement of the glomerular filtration surface, and (ii) restoration of the archetypical glomerular vascular pattern.
Glomerular capillaries of nephritic rats after combined VEGFR2 and PDGFRβ inhibition by PTK/ZK were tortuous and irregular. However, the onset of intussusceptive angiogenesis was influenced only after chronic PTK/ZK treatment providing an important insight into differential molecular regulation between sprouting and intussusceptive angiogenesis. PTK/ZK treatment abolished aSMA and tensin expression by injured mesangial cells, impaired glomerular filtration of microspheres and led to the reduction of glomerular volume and the presence of multiple hemorrhages detectable in the tubular system. Collectively, treatment of nephritic patients with PTK/ZK compound is not recommended.
Wnuk et al..Am J Pathol. 2011
PD Dr. Uyen Huynh-Do
Glomerular diseases starting with mesangiolysis have a high potential for recovery, previous work suggesting that podocytes could play a key role in the early phase of injury. The molecular mechanisms remain however to be defined. Eph receptor tyrosine kinases and their ligands (ephrins) play a pivotal role in the homeostasis of many adult organs and are also widely expressed in the kidney. The aim of our study was thus to assess their role in the glomerular recovery from Thy1.1 nephritis. EphB4 and ephrinBs were expressed in healthy glomerular podocytes and upregulated during Thy1.1 nephritis. Importantly, EphB4 was strongly phosphorylated around day 9 of nephritis. We therefore applied NPV-BHG712, an inhibitor of EphB4 phosphorylation, to control or nephritic rats. NPV-BHG712 induced no glomerular changes in controls. Nephritic animals treated with vehicle showed neither morphological evidence of podocyte injury nor podocyte loss. In contrast, NPV-BHG712 application to nephritic rats induced glomerular microaneurysms, podocyte damage and loss. Prolonged NPV-BHG712 treatment resulted in increased albuminuria and dysregulated mesangial recovery. Additionally, NPV-BHG712 inhibited capillary repair by intussusceptive angiogenesis, suggesting a previously unrecognized role of podocytes in regulating intussusceptive vessel splitting. In aggregate, our results identify EphB4 signalling as a novel pathway allowing podocytes to survive the transient capillary collapse.
Wnuk et al., Kidney International