[Show abstract][Hide abstract] ABSTRACT: The contribution of microglia to ischemic cortical stroke is of particular therapeutic interest because of the impact on the survival of brain tissue in the ischemic penumbra, a region that is potentially salvable upon a brain infarct. Whether or not tissue in the penumbra survives critically depends on blood flow and vessel perfusion. To study the role of microglia in cortical stroke and blood vessel stability, CX3CR1+/GFP mice were subjected to transient middle cerebral artery occlusion and then microglia were investigated using time-lapse two-photon microscopy in vivo. Soon after reperfusion, microglia became activated in the stroke penumbra and started to expand cellular protrusions towards adjacent blood vessels. All microglia in the penumbra were found associated with blood vessels within 24 h post reperfusion and partially fully engulfed them. In the same time frame blood vessels became permissive for blood serum components. Migration assays in vitro showed that blood serum proteins leaking into the tissue provided molecular cues leading to the recruitment of microglia to blood vessels and to their activation. Subsequently, these perivascular microglia started to eat up endothelial cells by phagocytosis, which caused an activation of the local endothelium and contributed to the disintegration of blood vessels with an eventual break down of the blood brain barrier. Loss-of-microglia-function studies using CX3CR1GFP/GFP mice displayed a decrease in stroke size and a reduction in the extravasation of contrast agent into the brain penumbra as measured by MRI. Potentially, medication directed at inhibiting microglia activation within the first day after stroke could stabilize blood vessels in the penumbra, increase blood flow, and serve as a valuable treatment for patients suffering from ischemic stroke.
[Show abstract][Hide abstract] ABSTRACT: Angiogenesis, defined as blood vessel formation from a preexisting vasculature, is governed by multiple signal cascades including integrin receptors, in particular integrin α(v)β(3). Here we identify the endothelial cell (EC)-secreted factor epidermal growth factor-like protein 7 (EGFL7) as a novel specific ligand of integrin α(v)β(3) thus providing mechanistic insight into its proangiogenic actions in vitro and in vivo. Specifically, EGFL7 attaches to the ECM and by its interaction with integrin α(v)β(3) increases the motility of EC, which allows EC to move on a sticky underground during vessel remodeling. We provide evidence that the deregulation of EGFL7 in zebrafish embryos leads to a severe integrin-dependent malformation of the caudal venous plexus (CVP), pointing towards the significance of EGFL7 in vessel development. In biopsies of patients with neurological diseases, vascular EGFL7 expression rose with increasing EC proliferation. Further, EGFL7 became upregulated in vessels of the stroke penumbra using a mouse model of reversible middle cerebral artery occlusion (MCAO). Our data suggest that EGFL7 expression depends on the remodeling state of the existing vasculature rather than on the phenotype of neurological disease analyzed. In sum, our work sheds a novel light on the molecular mechanism EGFL7 engages to govern physiological and pathological angiogenesis.
[Show abstract][Hide abstract] ABSTRACT: Deleted in malignant brain tumors 1 (DMBT1) belongs to the scavenger receptor cysteine-rich superfamily of proteins and is implicated in innate immunity, cell polarity, and differentiation. Here we studied the role of DMBT1 in endothelial cells.
DMBT1 was secreted into the extracellular matrix (ECM) by endothelial cells in vitro and in situ and the presence of DMBT1 in the ECM increased endothelial cell adherence. Endothelial cell-derived DMBT1 associated with galectin-3 (coprecipitation), and human recombinant DMBT1 bound EGF, vascular endothelial growth factor and Delta-like (Dll) 4 (specific ELISAs). Compared to cells from wild-type mice, endothelial cells from DMBT1(-/-) mice demonstrated reduced migration, proliferation, and tube formation. In vivo recovery from hindlimb ischemia was attenuated in DMBT1(-/-) animals as was vascular endothelial growth factor -induced endothelial sprouting from isolated aortic rings; the latter response could be rescued by the addition of recombinant DMBT1. The Notch pathway is involved in multiple aspects of vascular development, including arterial-venous differentiation and we found that endothelial cells from DMBT1(-/-) mice expressed more EphrinB2 than cells from wild-type mice. Levels of Dll1, Dll4, Hes1, Hey1, and EphB4, on the other hand, were decreased.
Taken together, the results of this study indicate that DMBT1 functions as an important endothelium-derived ECM protein that is able to bind angiogenic factors and promote adhesion, migration, proliferation, and angiogenesis as well as vascular repair. Mechanistically, DMBT1 interacts with galectin-3 and modulates the Notch signaling pathway as well as the differential expression of ephrin-B2 and EphB4.
[Show abstract][Hide abstract] ABSTRACT: Notch signalling is a key intercellular communication mechanism that is essential for cell specification and tissue patterning, and which coordinates critical steps of blood vessel growth. Although subtle alterations in Notch activity suffice to elicit profound differences in endothelial behaviour and blood vessel formation, little is known about the regulation and adaptation of endothelial Notch responses. Here we report that the NAD(+)-dependent deacetylase SIRT1 acts as an intrinsic negative modulator of Notch signalling in endothelial cells. We show that acetylation of the Notch1 intracellular domain (NICD) on conserved lysines controls the amplitude and duration of Notch responses by altering NICD protein turnover. SIRT1 associates with NICD and functions as a NICD deacetylase, which opposes the acetylation-induced NICD stabilization. Consequently, endothelial cells lacking SIRT1 activity are sensitized to Notch signalling, resulting in impaired growth, sprout elongation and enhanced Notch target gene expression in response to DLL4 stimulation, thereby promoting a non-sprouting, stalk-cell-like phenotype. In vivo, inactivation of Sirt1 in zebrafish and mice causes reduced vascular branching and density as a consequence of enhanced Notch signalling. Our findings identify reversible acetylation of the NICD as a molecular mechanism to adapt the dynamics of Notch signalling, and indicate that SIRT1 acts as rheostat to fine-tune endothelial Notch responses.
[Show abstract][Hide abstract] ABSTRACT: Despite extensive investigations of Cbl-interacting protein of 85 kDa (CIN85) in receptor trafficking and cytoskeletal dynamics, little is known about its functions in vivo. Here, we report the study of a mouse deficient of the two CIN85 isoforms expressed in the central nervous system, exposing a function of CIN85 in dopamine receptor endocytosis. Mice lacking CIN85 exon 2 (CIN85(Deltaex2)) show hyperactivity phenotypes, characterized by increased physical activity and exploratory behaviour. Interestingly, CIN85(Deltaex2) animals display abnormally high levels of dopamine and D2 dopamine receptors (D2DRs) in the striatum, an important centre for the coordination of animal behaviour. Importantly, CIN85 localizes to the post-synaptic compartment of striatal neurons in which it co-clusters with D2DRs. Moreover, it interacts with endocytic regulators such as dynamin and endophilins in the striatum. Absence of striatal CIN85 causes insufficient complex formation of endophilins with D2DRs in the striatum and ultimately decreased D2DR endocytosis in striatal neurons in response to dopamine stimulation. These findings indicate an important function of CIN85 in the regulation of dopamine receptor functions and provide a molecular explanation for the hyperactive behaviour of CIN85(Deltaex2) mice.
The EMBO Journal 07/2010; 29(14):2421-32. · 10.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Soluble components of Notch signalling can be applied to manipulate a central pathway essential for the development of metazoans and often deregulated in illnesses such as stroke, cancer or cardiovascular diseases. Commonly, the Notch cascade is inhibited by small compound inhibitors, which either block the proteolysis of Notch receptors by gamma-secretases or interfere with the transcriptional activity of the Notch intracellular domain. Specific antibodies can also be used to inhibit ligand-induced activation of Notch receptors. Alternatively, naturally occurring endogenous inhibitors of Notch signalling might offer a specific way to block receptor activation. Examples are the soluble variants of the canonical Notch ligand Jagged1 and the non-canonical Notch ligand Dlk1, both deprived of their transmembrane regions upon ectodomain shedding, or the bona fide secreted molecule EGFL7. We present frequently used methods to decrease Notch signalling, and we discuss how soluble Notch inhibitors may be used to treat diseases.
[Show abstract][Hide abstract] ABSTRACT: EGFL7 drives the formation of neurons from neural stem cells. In the embryonic and adult brain this process is essential for neurogenesis and homeostasis of the nervous system. The function of adult neurogenesis is not fully understood but maybe it supports life-long learning and brain repair after injuries such as stroke. The transition of neural stem cells into mature neurons is tightly regulated. One of the essential signaling pathways governing this process is the Notch pathway, which controls metazoan development. In a recent publication, we identified a novel non-canonical Notch ligand, EGFL7, and described its impact on neural stem cells. We explored the molecular mechanisms, which this molecule affects to regulate the self-renewal capacity of neural stem cells and to promote their differentiation into neurons. In this review, we discuss the implications of our findings for adult neurogenesis and illustrate the potential of EGFL7 to serve as an agent to increase neurogenesis and the self-renewal potential of the brain
[Show abstract][Hide abstract] ABSTRACT: Cancer progression is characterized by autarky in growth signals, insensitivity to growth-restrictive signals, evasion of apoptosis, a limitless potential to replicate, sustained angiogenesis, and tissue invasion, including metastasis. The regulation of these cellular processes relies on a fine-tuned control of molecular signal cascades. In recent years, short noncoding RNAs termed microRNAs (miRNAs) have been described as a novel class of molecular regulators. These affect various signaling cascades during the progression of neoplastic diseases by the regulation of gene expression on the post-transcriptional level. The novel endothelial cell-derived secreted protein epidermal growth factor-like domain 7 (EGFL7) has been suggested to control vascular tubulogenesis. Further, the two biologically active miRNAs miR-126 and its complement miR-126*, which are encoded by intron 7 of the egfl7 gene, have been described to mediate vascular functions. Knock-out studies in zebrafish and mice suggested a major role of miR-126 in angiogenesis and vascular integrity, which was mediated by the repression of inhibitors of VEGF-induced proliferation in endothelial cells. Recent studies revealed the distribution and function of miR-126 and miR-126* in various types of cancer, and assigned a role to both miRNAs as suppressors of tumor formation. Indeed, miR-126 and miR-126* have been reported to impair cancer progression through signaling pathways that control tumor cell proliferation, migration, invasion, and survival. Conversely, miR-126 and miR-126* may have a supportive role in the progression of cancer as well, which might be mediated by the promotion of blood vessel growth and inflammation. In this work, we will summarize the current knowledge on functions of miR-126/miR-126* that are relevant for cancer formation, and we will discuss their potential clinical use as predictive markers of survival and application as novel therapeutic targets for the treatment of neoplastic diseases.
The Scientific World Journal 01/2010; 10:2090-100. · 1.22 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Wnt/beta-catenin signaling has been implicated in taste papilla development; however, its role in epithelial maintenance and tumor progression in the adult tongue remains elusive. We show Wnt/beta-catenin pathway activation in reporter mice and by nuclear beta-catenin staining in the epithelium and taste papilla of adult mouse and human tongues. beta-Catenin activation in APC(min/+) mice, which carry a mutation in adenomatous poliposis coli (APC), up-regulates Sonic hedgehog (Shh) and Jagged-2 (JAG2) in the tongue epithelium without formation of squamous cell carcinoma (SCC). We demonstrate that Shh suppresses beta-catenin transcriptional activity in a signaling-dependent manner in vitro and in vivo. A similar regulation and function was observed for JAG2, suggesting that both pathways negatively regulate beta-catenin, thereby preventing SCC formation in the tongue. This was supported by reduced nuclear beta-catenin in the tongue epithelium of Patched(+/-) mice, exhibiting dominant active Shh signaling. At the invasive front of human tongue cancer, nuclear beta-catenin and Shh were increased, suggesting their participation in tumor progression. Interestingly, Shh but not JAG2 was able to reduce beta-catenin signaling in SCC cells, arguing for a partial loss of negative feedback on beta-catenin transcription in tongue cancer. We show for the first time that the putative Wnt/beta-catenin targets Shh and JAG2 control beta-catenin signaling in the adult tongue epithelium, a function that is partially lost in lingual SCC.
American Journal Of Pathology 01/2010; 177(1):404-414. · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Epidermal growth factor-like domain 7 (EGFL7) is a secreted factor implicated in cellular responses such as cell migration and blood vessel formation; however the molecular mechanisms underlying the effects of EGFL7 are largely unknown. Here we have identified transmembrane receptors of the Notch family as EGFL7-binding molecules. Secreted EGFL7 binds to a region in Notch involved in ligand-mediated receptor activation, thus acting as an antagonist of Notch signalling. Expression of EGFL7 in neural stem cells (NSCs)
[Show abstract][Hide abstract] ABSTRACT: Binding of epidermal growth factor (EGF) to its receptor leads to receptor dimerization, assembly of protein complexes, and activation of signaling networks that control key cellular responses. Despite their fundamental role in cell biology, little is known about protein complexes associated with the EGF receptor (EGFR) before growth factor stimulation. We used a modified membrane yeast two-hybrid system together with bioinformatics to identify 87 candidate proteins interacting with the ligand-unoccupied EGFR. Among them was histone deacetylase 6 (HDAC6), a cytoplasmic lysine deacetylase, which we found negatively regulated EGFR endocytosis and degradation by controlling the acetylation status of alpha-tubulin and, subsequently, receptor trafficking along microtubules. A negative feedback loop consisting of EGFR-mediated phosphorylation of HDAC6 Tyr(570) resulted in reduced deacetylase activity and increased acetylation of alpha-tubulin. This study illustrates the complexity of the EGFR-associated interactome and identifies protein acetylation as a previously unknown regulator of receptor endocytosis and degradation.
[Show abstract][Hide abstract] ABSTRACT: Sustained growth of solid tumours can rely on both the formation of new and the co-option of existing blood vessels. Current models suggest that binding of angiopoietin-2 (Ang-2) to its endothelial Tie2 receptor prevents receptor phosphorylation, destabilizes blood vessels, and promotes vascular permeability. In contrast, binding of angiopoietin-1 (Ang-1) induces Tie2 receptor activation and supports the formation of mature blood vessels covered by pericytes. Despite the intense research to decipher the role of angiopoietins during physiological neovascularization and tumour angiogenesis, a mechanistic understanding of angiopoietin function on vascular integrity and remodelling is still incomplete. We therefore assessed the vascular morphology of two mouse mammary carcinoma xenotransplants (M6378 and M6363) which differ in their natural angiopoietin expression. M6378 displayed Ang-1 in tumour cells but no Ang-2 in tumour endothelial cells in vivo. In contrast, M6363 tumours expressed Ang-2 in the tumour vasculature, whereas no Ang-1 expression was present in tumour cells. We stably transfected M6378 mouse mammary carcinoma cells with human Ang-1 or Ang-2 and investigated the consequences on the host vasculature, including ultrastructural morphology. Interestingly, M6378/Ang-2 and M6363 tumours displayed a similar vascular morphology, with intratumoural haemorrhage and non-functional and abnormal blood vessels. Pericyte loss was prominent in these tumours and was accompanied by increased endothelial cell apoptosis. Thus, overexpression of Ang-2 converted the vascular phenotype of M6378 tumours into a phenotype similar to M6363 tumours. Our results support the hypothesis that Ang-1/Tie2 signalling is essential for vessel stabilization and endothelial cell/pericyte interaction, and suggest that Ang-2 is able to induce a switch of vascular phenotypes within tumours.
The Journal of Pathology 12/2008; 217(4):571-80. · 7.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Ataxin-2 is a novel protein, where the unstable expansion of an internal polyglutamine domain can cause the neurodegenerative disease Spinocerebellar Ataxia type 2 (SCA2). To elucidate its cellular function, we have used full-length ataxin-2 as bait in a yeast two-hybrid screen of human adult brain cDNA. As binding partners we found endophilin A1 and A3, two brain-expressed members of the endophilin A family involved in synaptic vesicle endocytosis. Co-immunoprecipitation studies confirmed the binding of these proteins as an endogenous complex in mouse brain. In vitro binding experiments narrowed the binding interfaces down to two proline-rich domains on ataxin-2, which interacted with the SH3 domain of endophilin A1/A3. Ataxin-2 and endophilin associated at the endoplasmic reticulum as well as at the plasma membrane as determined by immunofluorescence microscopy of transfected cell lines, and by centrifugation fractionation studies of mouse brain. Importantly, the pattern observed in transfected cells was conserved in rat hippocampal neurons. In the mouse brain, an association of ataxin-2 with endocytic proteins such as the adaptor CIN85 and the ubiquitin ligase c-Cbl was also demonstrated. GST pull-down assays showed ataxin-2 to directly interact with the SH3 domains A and C of CIN85 and with the SH3 domain of Src, a kinase activated after receptor stimulation. Functional studies demonstrated that ataxin-2 affects endocytic trafficking of the epidermal growth factor receptor (EGFR). Taken together, these data implicate ataxin-2 to play a role in endocytic receptor cycling.
[Show abstract][Hide abstract] ABSTRACT: Proteins of the Cbl family are adaptor molecules and ubiquitin ligases with major functions in the regulation, intracellular transport and degradation of receptor tyrosine kinases (RTKs). Due to this central role, mutations that cause malfunctions of Cbl or their associated proteins - termed the Cbl interactome - easily lead to the transformation of affected cells and eventually the development of cancer. This review intends to give an overview on the mechanisms of Cbl-mediated cell transformation in light of the dysregulated intracellular trafficking of RTKs.
European Journal of Cell Biology 10/2007; 86(9):505-12. · 3.70 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In acute myeloid leukemia (AML), mutational activation of the receptor tyrosine kinase (RTK) Flt3 is frequently involved in leukemic transformation. However, little is known about a possible role of highly expressed wild-type Flt3 in AML. The proto-oncogene c-Cbl is an important regulator of RTK signaling, acting through its ubiquitin ligase activity and as a platform for several signaling adaptor molecules. Here, we analyzed the role of c-Cbl in Flt3 signal transduction and myeloid transformation. C-Cbl physically interacted with Flt3 and was tyrosine phosphorylated in the presence of Flt3-ligand (FL). Overexpression of a dominant-negative form of c-Cbl (Cbl-70Z) inhibited FL-induced Flt3 ubiquitylation and internalization, indicating involvement of c-Cbl in Flt3 signaling. DNA sequencing of AML bone marrow revealed a case with a c-Cbl point mutation (Cbl-R420Q). Cbl-R420Q inhibited Flt3 internalization and ubiquitylation. Coexpression of Cbl-R420Q or Cbl-70Z with Flt3 induced cytokine-independent growth and survival of 32Dcl3 cells in the absence of FL. Also, the mutant Cbl proteins altered the amplitude and duration of Flt3-dependent signaling events. Our results indicate an important role of Cbl proteins in Flt3 signal modulation. Also, the data suggest a novel mechanism of leukemic transformation in AML by mutational inactivation of negative RTK regulators.
[Show abstract][Hide abstract] ABSTRACT: Angiopoietins play important roles in the formation of neovessels and complex vascular networks. Angiopoietin (Ang)-1 and Ang-2 belong to a family of growth factors that display opposing effects on the activation of Tie2 (tyrosine kinase with immunoglobulin and epidermal growth factor homology domain 2). Endothelial Ang-2 expression is associated with vessel destabilization and regulates a balance between vascular regression and growth. To elucidate, in particular, the role of Ang-2 after arterial artery occlusion in the mouse limb, we applied a transgenic animal model with targeted Ang-2 expression in endothelial cells. We show here that restoration of blood flow in Ang-2:Tie1 transgenic mice is dramatically impaired when Ang-2 expression is induced in the vasculature. The defective restoration of perfusion in Ang-2 transgenic mice is evidenced by reduced collateral artery growth, which typically occurs to compensate for flow deficits after occlusion of the large conductance artery. Furthermore, reduced movement capacities and higher incidents of necrosis are consequently observed in the transgenic limbs as compared with controls. Mechanistically, the observed effects are attributed to defective smooth muscle cell recruitment in Ang-2 transgenic mice. Moreover, distinct Ang-2 levels in the genetically modified animals clearly correlated with the magnitude of reduced perfusion. In conclusion, our studies define Ang-2 as an important molecule for the progression of collateral artery growth and angiogenesis during ischemia and suggest precise Ang-2 dosage activities to accomplish blood vessel growth.
Circulation Research 08/2007; 101(1):88-96. · 11.09 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Ubiquitin (Ub) and ubiquitin-like (Ubl) proteins are small signaling molecules that are involved in many aspects of cell function. It has been assumed that Ub and Ubl have functionally distinct roles because they use different conjugation machineries and bind to different effector proteins. This paradigm, however, must be revisited after recent findings that signaling cascades mediated by Ub and the Ubl NEDD8 (Neural precursor cell-Expressed Developmentally Down-regulated 8) in the regulation of epidermal growth factor receptor (EGFR) endocytosis are redundant. In this context, Ub and NEDD8 share the same E3 ligase, Cbl, and are recognized by identical components of the endocytic sorting machinery. This unexpected redundancy introduces additional complexity to the current view of Ub signaling pathways.
[Show abstract][Hide abstract] ABSTRACT: Recently the life cycles of receptor tyrosine kinases (RTKs) have become a focus of signal transduction research. Ligand-induced ubiquitination of RTKs followed by their internalization and degradation has, in particular, been extensively studied. This chapter describes the basic methods used to measure ubiquitination and degradation of RTKs using the example of the epidermal growth factor receptor (EGFR). Common sources for endogenous and recombinant EGFR as well as cell lines used to conduct receptor downregulation assays are described. Monitoring of ubiquitination and degradation of the EGFR subsequent to stimulation with the receptor ligand EGF is described. Finally, protocols to quantitatively measure degradation of the EGFR by pulse chase experiments or using radiolabeled ligands such as 125I-EGF are presented.
[Show abstract][Hide abstract] ABSTRACT: Cbl proteins are ubiquitin ligases and multifunctional adaptor proteins that are implicated in the regulation of signal transduction in various cell types and in response to different stimuli. Cbl-associated proteins can assemble together at a given time or space inside the cell, and such an interactome can form signal competent networks that control many physiological processes. Dysregulation of spatial or temporal constraints in the Cbl interactome results in the development of human pathologies such as immune diseases, diabetes and cancer.
[Show abstract][Hide abstract] ABSTRACT: The ubiquitin ligase Cbl mediates ubiquitination of activated receptor tyrosine kinases (RTKs) and interacts with endocytic scaffold complexes, including CIN85/endophilins, to facilitate RTK endocytosis and degradation. Several mechanisms regulate the functions of Cbl to ensure the fine-tuning of RTK signalling and cellular homeostasis. One regulatory mechanism involves the binding of Cbl to Sprouty2, which sequesters Cbl away from activated epidermal growth factor receptors (EGFRs). Here, we show that Sprouty2 associates with CIN85 and acts at the interface between Cbl and CIN85 to inhibit EGFR downregulation. The CIN85 SH3 domains A and C bind specifically to proline-arginine motifs present in Sprouty2. Intact association between Sprouty2, Cbl and CIN85 is required for inhibition of EGFR endocytosis as well as EGF-induced differentiation of PC12 cells. Moreover, Sprouty4, which lacks CIN85-binding sites, does not inhibit EGFR downregulation, providing a molecular explanation for functional differences between Sprouty isoforms. Sprouty2 therefore acts as an inducible inhibitor of EGFR downregulation by targeting both the Cbl and CIN85 pathways.