Published online before print July 9, 2009, doi: 10.1161/CIRCRESAHA.109.195156
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© 2009 American Heart Association, Inc.
Cellular Biology |
Angiomotin-Like Protein 1 Controls Endothelial Polarity and Junction Stability During Sprouting Angiogenesis
From the Department of Oncology and Pathology (Y.Z., S.V., S.N., I.M., T.T., L.H.), Cancer Centrum Karolinska; and Laboratory of Molecular Neurobiology (P.U.) and Division of Matrix Biology (A.M.), Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden; and Institut National de la Santé et de la Recherche Médicale (S.A., J.-P.B.), U891, Centre de Recherche en Cancérologie de Marseille, Institut Paoli-Calmettes, Univ Méditerranée, Marseille, France.
Correspondence to Lars Holmgren, Department of Oncology and Pathology, Cancer Centrum Karolinska, Karolinska Institutet, SE-17176 Stockholm, Sweden. E-mail lars.holmgren{at}ki.se
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Abstract |
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Rationale: We have previously shown that angiomotin (Amot) is essential for endothelial cell migration during mouse embryogenesis. However,
5% of Amot knockout mice survived without any detectable vascular defects. Angiomotin-like protein 1 (AmotL1) potentially compensates for the absence of Amot as it is 62% homologous to Amot and exhibits similar expression pattern in endothelial cells. Objective: Here, we report the identification of a novel isoform of AmotL1 that controls endothelial cell polarization and directional migration.
Methods and Results: Small interfering RNA–mediated silencing of AmotL1 in mouse aortic endothelial cells caused a significant reduction in migration. In confluent mouse pancreatic islet endothelial cells (MS-1), AmotL1 colocalized with Amot to tight junctions. Small interfering RNA knockdown of both Amot and AmotL1 in MS-1 cells exhibited an additive effect on increasing paracellular permeability compared to that of knocking down either Amot or AmotL1, indicating both proteins were required for proper tight junction activity. Moreover, as visualized using high-resolution 2-photon microscopy, the morpholino-mediated knockdown of amotl1 during zebrafish embryogenesis resulted in vascular migratory defect of intersegmental vessels with strikingly decreased junction stability between the stalk cells and the aorta. However, the phenotype was quite distinct from that of amot knockdown which affected polarization of the tip cells of intersegmental vessels. Double knockdown resulted in an additive phenotype of depolarized tip cells with no or decreased connection of the stalk cells to the dorsal aorta.
Conclusions: These results cumulatively validate that Amot and AmotL1 have similar effects on endothelial migration and tight junction formation in vitro. However, in vivo Amot appears to control the polarity of vascular tip cells whereas AmotL1 mainly affects the stability of cell–cell junctions of the stalk cells.
Key Words: AmotL1 • polarity • migration • junction stability • zebrafish
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Introduction |
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The mechanisms of blood vessel development have received much attention because of their involvement in pathological processes such as neoplasia and ocular diseases.1–3 The major blood vessels, such as the aorta, are initially formed through the process of vasculogenesis in which precursor cells migrate, differentiate, and polarize to form tube-like structures.4 Once the central vessels are formed they extend, sprout and migrate to form a secondary capillary bed that supports the developing tissues with oxygen and nutrients. The migration trajectories are guided by attractive and repulsive signals that control the precise formation of a circulatory network in time and space.5 During angiogenesis, the leading endothelial cells (ECs), tip cells, extend filopodia that detect migratory cues in the microenvironment. The stalk cells form lumens and maintain cell–cell contacts to form a seamless vessel.6 Although a number of promigratory molecules have been identified little is yet known about how the extracellular signals exert their control on cell polarity, cell–cell junction stability and lumen formation.
Angiomotin (Amot) is a coiled-coil protein that is expressed in ECs and controls migration, tight junction (TJ) formation and cell polarity in vitro via its C-terminal PDZ-binding motif.7–9 Consistent with these in vitro findings, Amot-deficient mice of C57/B6-background showed severe vascular insufficiency in the intersomitic region as well as dilated vessels in the brain resulting in embryonic lethality between embryonic day (E)11 and E11.5.10 In vitro studies also revealed that Amot-deficient ECs lost polarity and had reduced directional migration in response to chemoattractants.10 In addition, DNA vaccination or antibody treatment against Amot significantly inhibited tumor angiogenesis and tumor growth, indicating that Amot is a potential target for cancer therapy.11,12 Despite the significant contribution of Amot to angiogenesis, it was noted that 5% of Amot-deficient mice with C57/B6 genetic background did survive without any detectable vascular defects.10 This led to the speculation that other members of the same protein family may compensate for the loss of Amot.
Amot together with angiomotin-like protein (AmotL)1 (AmotL1, also referred to as JEAP) and AmotL2 (also referred to as LCCP or MASCOT) belong to a novel protein family characterized by their conserved glutamine rich domain, coiled-coil domains and PDZ-binding motif.13 AmotL1 was initially cloned from MS-1 EC line as a TJ-enriched and associated protein (JEAP).14,15 However, the biological function of AmotL1 has yet to be characterized. Here, we report the identification of a novel isoform of AmotL1 that controls endothelial migration and cell polarity. We provide evidence that AmotL1 regulates sprouting angiogenesis by affecting tip cell migration as well as controlling cell–cell adhesions in vivo.
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An expanded Methods section is available in the Online Data Supplement at http://circres.ahajournals.org.
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Identification of the Short Isoform of AmotL1
In collaboration with the Human Protein Atlas consortium, we have analyzed AmotL1 protein expression in human tissue arrays. Immunohistochemical analysis showed that AmotL1 was detectable in ECs of human placenta, ovary, and cancer tissues (http://www.proteinatlas.org). We further examined the expression pattern of AmotL1 in EC lines including human umbilical vein ECs, bovine capillary ECs (BCEs), mouse pancreatic islet ECs (MS-1), mouse aortic ECs (MAEs), polyoma middle T ECs (derived from embryonic stem cells), and tumor ECs by Western blot. In addition to the band with a molecular mass more than 100 kDa, which has been reported as JEAP previously,14 we could detect another band with an apparent molecular mass of 90 kDa (Figure 1A).
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To assess the sequence of the 90-kDa band, AmotL1 was immunoprecipitated from MAE lysate and separated by SDS-PAGE. Two bands with respective molecular mass of 100 and 90 kDa were excised for mass spectrometric analysis. AmotL1 was unambiguously identified by Peptides Mass Fingerprint in both bands, indicating the existence of 2 AmotL1 isoforms. The N-terminal initiation site of the 90-kDa band was identified by Edman degradation (Figure 1B and Online Table I). Taken together, these data show that the 2 isoforms of AmotL1 only differ in the N-terminus. We subsequently cloned the short isoform from the DNA sequence corresponding to the 86th methionine of the original AmotL1 amino acid sequence (Figure 1C). Western blot analysis revealed that when transiently transfected into CHO cells, the cloned AmotL1 of 90 kDa was of the same molecular mass as the endogenous one from MAEs (Figure 1D). Thus, we denominated the 2 isoforms of AmotL1 as p90 and p100 AmotL1 according to their molecular mass.
AmotL1 Localizes to Endothelial Lamellipodia and Tight Junctions
Next, we investigated the subcellular localization of endogenous AmotL1 in ECs. Immunofluorescence staining of migrating MAEs showed that endogenous AmotL1 localized to the cell leading front edge lamellipodia and overlapped with F-actin as visualized by phalloidin staining, suggesting that AmotL1 might regulate EC migration (Figure 2A). In addition, AmotL1-positive staining was also detected in the cell–cell contacts of confluent MS-1 ECs where it colocalized with the TJ marker zonula occludens (ZO)-1 (Figure 2B). To further investigate whether Amot and AmotL1 colocalized to TJs, MS-1 cells were double stained with rat anti-mouse AmotL1 monoclonal antibody and rabbit anti-human Amot polyclonal antibodies. Immunofluorescence signals with the respective antibody showed perfect overlap in cell–cell junctions, indicating that the 2 proteins may interact directly with each other (Figure 2C).
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The PDZ-Binding Motifs Are Not Required for the Direct Interaction Between Amot and AmotL1
We have previously reported that Amot and AmotL1 bind to the scaffold protein Patj (or Mupp1) through distinct PDZ domain and PDZ-binding motif, which opens up the possibility that Amot and AmotL1 could be part of the same protein complex.9 However, there is no evidence that Amot and AmotL1 directly bind to each other. All of the members in the angiomotin family share the conserved coiled-coil domains, through which they form homooligomers.16,17 Based on their colocalization in ECs, it might be speculated that Amot and AmotL1 could either form heterooligomers through the conserved coiled-coil domains or by binding to distinct PDZ domains of Patj (or Mupp1) to stabilize the polarity protein complex Amot:AmotL1:Patj. To verify this hypothesis, p80 Amot and p100 AmotL1 or their mutants at PDZ-binding motif (
PDZ) were cotransfected into CHO cells, which lack endogenous expression of both Amot and AmotL1. The immunoprecipitation results revealed that Amot and AmotL1 coprecipitated with each other independent of whether the PDZ-binding motifs were intact or not (Figure 3). Thus, Amot and AmotL1 can form heterooligomers most likely through the direct binding of coiled-coil domains and their PDZ-binding motifs are not required during the direct interaction between Amot and AmotL1.
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AmotL1 Controls Cell Polarity and Promotes Endothelial Cell Migration
To investigate the biological functions of AmotL1, SMARTpool small interfering (si)RNA specifically targeting AmotL1 was transfected into MAEs (which only express AmotL1; Figure 1A). Western blot analysis confirmed that both p90 and p100 AmotL1 were efficiently knocked down by using siRNA (Figure 4A). Cell polarity was studied by analyzing Golgi positioning.18,19 Reorientation of the Golgi is thought to facilitate polarized secretion thereby providing membrane and secreted products directly to the most proximate plasma membrane such as the leading edge in migrating cells.20 Visualization of Golgi apparatus with a GM130 antibody revealed that the Golgi apparatus of most AmotL1-silenced MAEs surrounded the nucleus, whereas in the majority of control siRNA-treated MAEs the Golgi located within one 120° sector relative to the nucleus (Figure 4B). Compared to that transfected with control siRNA, the polarity of AmotL1 siRNA-treated MAEs showed 50% reduction in Golgi reorientation (Figure 4C).
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Because the polarization of the cell is related to the directional migration, we further examined the migration of AmotL1 siRNA-treated MAEs with the Boyden chamber assay. The results indicated that both basal and fibroblast growth factor (FGF)2-induced migration were significantly decreased when the MAEs were treated with AmotL1 siRNA for 72 hours (Figure 4D). We then constructed MAE lines stably expressing the p90 or p100 AmotL1 isoforms. Stable expression of the p100 AmotL1 in MAEs also resulted in overexpression of p90 AmotL1 as compared to the control cell line transfected with vector alone (Figure 4E). Consistent with our findings that AmotL1 controls Golgi positioning, p90 and p100 AmotL1 MAEs exhibited increased polarity (Figure 4F and 4G) and upregulated migration toward FGF2 in comparison with MAE vector cells. However, p90 AmotL1 MAEs possessed an even higher migratory attributable to the expression level of p90 AmotL1 (Figure 4H).
AmotL1 Regulates Paracellular Permeability
The results of the immunofluorescence staining demonstrated that stably transfected p90 and p100 AmotL1 localized to cell–cell junctions in the confluent CHO cells (Figure 5A). Although CHO cells do not form properly organized TJs, they are commonly used in an in vitro permeability assay to study the function of proteins, which may promote TJ formation. According to their junctional localization in CHO cells, we hypothesized that both AmotL1 isoforms might affect TJ function and paracellular permeability. To verify this hypothesis, CHO cells stably transfected with p100 and p90 AmotL1 were used in an in vitro paracellular permeability assay where the diffusion of fluorescein isothiocyanate (FITC)-labeled dextran across a monolayer of cells grown on a permeable membrane was measured. FITC-dextran was added to the upper chamber and aliquots from the bottom chamber were removed for quantitation of the FITC-dextran by fluorimetry. A significant difference in monolayer permeability was observed between CHO vector and AmotL1-transfected CHO cells. The diffusion of FITC-dextran to the bottom chamber gradually increased from p90 or p100 AmotL1 CHO cells monolayer which was comparable to that of p130 Amot and after 8 hours, the fluorescence intensity was 50% reduced compared with that of CHO vector cells (Figure 5B). In contrast, using the same approach, we also found that the paracellular permeability of Amot or AmotL1 siRNA-treated MS-1 ECs significantly increased compared with that of control siRNA-treated MS-1 cells. Double knockdown of Amot and AmotL1 via siRNA resulted in an additive effect on increasing the paracellular permeability of MS-1 cells (Figure 5C and 5D). These results indicated that Amot and AmotL1 not only colocalized at the cell–cell junctions but also regulated proper TJ activity.
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Knockdown of Zebrafish amotl1 Causes Vascular Deficiency During Embryogenesis
The zebrafish is an important vertebrate model for the studies of gene functions in vivo because of its unique advantages, such as large numbers of embryos, rapid and external embryonic development, and optical clarity, which allows the direct visualization of organogenesis.21,22 We identified the zebrafish amotl1 ortholog by BLAST searches against the Ensembl and NCBI databases. Zebrafish amotl1 is present in one copy on chromosome 15 and shows overall 48% amino acid identity with human AmotL1 (Online Figure I). During embryogenesis, the zebrafish fli1 gene is expressed in the hemangioblast and ECs.23 To address the expression of amot and amotl1 in vivo, fli1:EGFP transgenic zebrafish in which green fluorescent protein (GFP) was expressed in the forming vasculature was used for collecting GFP+ cells at 26 hours postfertilization (hpf) by fluorescence-activated cell sorting (Figure 6A). Zebrafish amot and amotl1 expression levels were examined by quantitative real-time polymerase chain reaction (PCR) with the standard curve method (User Bulletin 2, Applied Biosystems, 2001). The results revealed that similar to zebrafish endothelial-specific gene VE-cadherin (CDH5), the expression of both amot and amotl1 were 9-fold increased in GFP+ ECs as compared to the unsorted control (Figure 6B). This indicates that during early stage of zebrafish development amot and amotl1 are primarily expressed in vascular ECs.
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To determine whether amotl1 was required for vessel development in vivo, 3 different antisense morpholinos (MOs) targeting exon 6, exon 10, or exon 12 of zebrafish amotl1 were individually injected into embryos of fli1:EGFP transgenic zebrafish at the 1 to 2 cell stage. None of the injected-MOs induced any gross morphological defects as examined under bright field microscopy (Online Figure II). The injection of 3 different amotl1 MOs resulted in similar vascular defects in intersegmental vessels (ISVs) migration (Online Figure III). We then focused on MO1 and the knockdown efficiency was confirmed by RT-PCR at 30 hpf. Injection of amotl1 MO1 caused the appearance of shorter mRNAs as detected by RT-PCR, demonstrating that the amotl1 transcript was affected in these morphants (Figure 7A). Sequencing and conceptual translation of the RT-PCR products showed that both amotl1 morphant mRNAs encoded the prematurely truncated proteins (Figure 7B and Online Figure IV).
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Compared to zebrafish treated with the mismatched control MO1 at 32 hpf, amotl1 morphants exhibited defective ISV migration and most of the ISVs were typically arrested at the horizontal myoseptum, a phenotype shared in common with the amot morphants. However, in comparison to the phenotype of amot morphants, the penetrance and severity of the amotl1 phenotype were lower (Figure 7C and 7E). To determine whether amot and amotl1 have overlapping functions during endothelial migration, we simultaneously knocked down both amot and amotl1. Quantification revealed that injection of amotl1 MO1 caused 33% of zebrafish embryos with ISV defective phenotype at 40 hpf and the average number of defective ISVs per embryo was 4.2±1.8. Whereas coinjection of amot and amotl1 MOs resulted in 78% of the embryos showing an ISV phenotype at 40 hpf. The average number of defective ISVs per embryo with the dual knockdown was increased to 12±3.1, which was significantly higher than that of either amot or amotl1 single MO injection. The results indicated that amot and amotl1 had overlapping roles in controlling EC migration and angiogenesis (Figure 7C and 7F).
To further verify that the defect in ISV migration was specifically caused by amotl1 knockdown, as well as further validate the potential overlap function between amot and amotl1, rescue experiments were carried out with the coinjection of amotl1 MO1/mouse p90 AmotL1 mRNA, amotl1 MO1/mouse p100 AmotL1 mRNA, or amotl1 MO1/human p80 Amot mRNA, respectively. As shown in Figure 7G, at 36 hpf, both p90 mAmotL1 and p80 hAmot mRNA had very similar effects in rescuing the ISV defective phenotype of amotl1 morphants. In contrast, p100 mAmotL1 mRNA could not rescue the ISV migration defects. These results demonstrated that the vascular phenotype was specifically caused by amotl1 gene knockdown and provided additional substantiation that primarily the p90 isoform of AmotL1 promotes EC migration.
Examination with high-resolution 2-photon microscopy revealed distinct differences of ISV defects between amot and amotl1 morphants. When amot was knocked down, the tip cells of ISVS exhibited a depolarized phenotype, spread horizontally, and extended filopodia in an unpolarized manner (Figure 7D and Online Movie II). In contrast, the ISVs of amotl1 morphants became arrested at the horizontal myoseptum and the polarity of some tip cells was also affected. However, the striking and distinct phenotype of amotl1 morphants was that the cell–cell junctions between stalk cells and the dorsal aorta appeared perturbed (Figure 7D and Online Movie III). In addition, the double knockdown of both amot and amotl1 combined these phenotypes with depolarized tip cells as well as inhibited connection to the aorta (Figure 7D and Online Movie IV).
The endothelial junctional defect of amotl1 morphants was further visualized by whole mount immunofluorescence staining of claudin-5. The claudin-5 together with the GFP signal of ISVs from control fish showed stable connection of stalk cell to the dorsal aorta (Figure 8A, left), whereas weaker connection between ISV stalk cells and the dorsal aorta was detected in amotl1 morphants (Figure 8A, right). The zebrafish ISV is composed of 3 ECs by 30 hpf. The tip cell (designated as No.3 EC in Figure 8A), stalk cell (No.2 EC), and inverted T-shaped EC (No.1 EC).24,25 Because of the procedures of claudin-5 whole mount immunofluorescence staining, the GFP signal in the cytoplasm of zebrafish vascular EC became weaker than that in the nucleus, which allowed us to visualize the nuclei of 3 different ISV ECs. As shown in Figure 8A, both the tip cell and stalk cell in the ISV of amotl1 morphant stayed at the horizontal myoseptum, indicating the tip cell migration was halted compared to that of the control ISVS. In addition, we confirm that the cell–cell junction defect of amotl1 morphant is between stalk cell and inverted T-shaped EC by the combination of visualization of EC nucleus, GFP signal, and claudin-5 staining.
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Discussion |
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Here, we report the characterization of a novel isoform of AmotL1 which controls endothelial migration and TJ formation in vitro. We further show that AmotL1 and Amot have overlapping but distinct roles in vertebrate angiogenesis in vivo.
We have previously shown that angiomotin is essential for directional migration of ECs by the formation of a signaling scaffold.9 This signaling complex consists in part of the Pals:Patj:Lin7 polarity proteins that are evolutionary conserved and promotes cell polarity in epithelial cells. In ECs, this complex is also involved in determining cell polarity of migrating tip cells, as judged by the lack of polarized extensions of filopodia in vivo.10 In this report, we have studied the roles of AmotL1 in blood vessel formation. Because AmotL1, like Amot, associates to the Pals:Patj:Lin7 polarity complex, we propose that AmotL1 promotes migration by controlling cell polarity. One possible effector may be the RhoGEF Syx (PLEKHG5/TECH) that regulates RhoA activity in a spatiotemporal manner in ECs and forms a ternary complex with AmotL1 via the interaction of Mupp1/Patj.9
AmotL1 was initially identified as a TJ enriched protein consisting of the coiled-coil domains and PDZ-binding motif.14 Coiled-coil domains have been identified in a variety of cytoskeletal proteins and are involved in inter- or intramolecular protein–protein interactions. Indeed, all members of the Amot family have been shown to oligomerize via the coiled-coil domains. Here, we further show that Amot and AmotL1 could form heterooligomers and colocalized in endothelial TJs in vitro. Both proteins interact with individual PDZ domains of Patj/Mupp1, but this interaction is not required for oligomerizations because Amot:AmotL1 formed a complex even when the C-terminal PDZ-binding motifs were mutated. Whether the Amot:AmotL1 heterooligomers activate different signaling pathways than the homooligomers remains to be shown. Functional analysis of Amot and AmotL1 in TJ formation showed that exogenously overexpression of both proteins decreases paracellular permeability. Interestingly, siRNA knockdown of both Amot and AmotL1 in MS-1 ECs exhibited an additive effect on paracellular permeability, suggesting that both proteins may be required for proper TJ activity in ECs.
The dorsal aorta of zebrafish is derived from angioblasts migration out of the lateral plate mesoderm, and this process is an early event in vasculogenesis.26–28 The phenotypes of zebrafish amotl1 knockdown showed that the formation of dorsal aorta and the subsequent ECs sprouting from dorsal aorta to the horizontal myoseptum were not affected, suggesting that amotl1 is not required during vasculogenesis. In contrast, the endothelial tip cell migration from the horizontal myoseptum to the dorsal neural tube was greatly hindered when zebrafish amotl1 was knocked down, indicating that amotl1 is essential for ECs migration during sprouting angiogenesis. However, the ISV sprouting from dorsal aorta to the horizontal myoseptum occurred in an amotl1-independent manner. This might be attributable to the existence of alternative signaling pathway during this period or the expression of another protein with a function analogous to amotl1 but only active before the expression of functional amotl1.
There appeared to be a distinct difference between the vessels that were arrested because of either amot or amotl1 knockdown. When amot expression was inhibited, the sprouting tip cells was arrested and spread horizontally along the myoseptum and extended filopodia in a multi-directional manner (Figure 7D). In amotl1 knockdown vessels, sprouting vessels were arrested but horizontal spreading of tip cells was less observed than amot knockdown. However, the connections between the stalk cells and the aorta appeared to be destabilized as observed by high-resolution 2-photon imaging, time-lapse photography, and visualizing endothelial junctions with claudin-5 staining. The double knockdown phenotype adds these 2 phenotypes together as the tip cells appeared depolarized and the connection to the aorta is destabilized (Figure 7D and Online Movie IV).
There are several possible explanations for why amot and amotl1 exert different effects on ISV formation. Firstly, the spatiotemporal expression pattern may differ. This has been shown by analysis of protein expression during different stages of retinal angiogenesis in newborn mice (Y Zheng, unpublished data, 2009).16 Secondly, both Amot and AmotL1 are associated to the Patj/Mupp1 scaffold proteins. However, these protein complexes differ in that Pals2, filamin A, and PTN13 are associated to Amot but not to the other proteins of the Amot family.9 It is also possible that level oligomerization may locally affect signal in the developing vessels.
In conclusion, our findings show that AmotL1 functions as a key regulator of EC migration and cell–cell junction stability during zebrafish embryogenesis. The results of this study emphasize the role of AmotL1 in angiogenesis, and we speculate that the combination of Amot and AmotL1 might be a potential future targets for antiangiogenic therapy.
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Acknowledgments |
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We thank Susan Warner, Ulla Wargh, and Sajila Kisana from the Karolinska Institute zebrafish facility for experimental support. We thank ZIRC for providing zebrafish. We are very grateful to Dr Yoshimi Takai for kindly providing JEHP monoclonal antibody.
Sources of Funding
This work was supported by grants from the Swedish Cancer Society, the Cancer Society of Stockholm, the Swedish Research Council, EUCAAD, Karolinska Institutet (to L.H.); and Knut och Alice Wallenbergs Stiftelse (CLICK) and a postdoctoral stipend from the Wenner-Gren Foundation (to Y.Z.). J.-P.B. is supported by EUCAAD, La Ligue Contre le Cancer (Label 2007), Institut Paoli-Calmettes, INCa, and IBISa (Marseille Proteomic Platform; http://map.univmed.fr).
Disclosures
None.
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Footnotes |
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*Both authors contributed equally to this work.
Original received February 14, 2009; revision received June 28, 2009; accepted June 29, 2009.
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