Mechanisms underlying the vein development remain largely unknown. Tie2 signaling mediates endothelial cell (EC) survival and vascular maturation and its activating mutations are linked to venous malformations. Here we show that vein formation are disrupted in mouse skin and mesentery when Tie2 signals are diminished by targeted deletion of Tek either ubiquitously or specifically in embryonic ECs. Postnatal Tie2 attenuation resulted in the degeneration of newly formed veins followed by the formation of haemangioma-like vascular tufts in retina and venous tortuosity. Mechanistically, Tie2 insufficiency compromised venous EC identity, as indicated by a significant decrease of COUP-TFII protein level, a key regulator in venogenesis. Consistently, angiopoietin-1 stimulation increased COUP-TFII in cultured ECs, while Tie2 knockdown or blockade of Tie2 downstream PI3K/Akt pathway reduced COUP-TFII which could be reverted by the proteasome inhibition. Together, our results imply that Tie2 is essential for venous specification and maintenance via Akt mediated stabilization of COUP-TFII.https://doi.org/10.7554/eLife.21032.001
Mechanisms underlying arteriovenous specification have been under intensive investigation during the past years, and this has led to the identification of several signaling pathways involved in the coordination of this process. The VEGF-A/VEGFR-2 pathway mediates activation of RAF1 and ERK1/2 kinases to induce the expression of genes required for arterial development (Lanahan et al., 2013; Deng et al., 2013), including Delta-like 4 (Dll4) that activates NOTCH signaling (Lawson et al., 2001; Duarte et al., 2004; Wythe et al., 2013). Wnt/β-catenin, SOX17 and FOXC1/2 were also reported to participate in arterial development via activation of the NOTCH pathway (Seo et al., 2006; Corada et al., 2010, 2013). In contrast, knowledge on venogenesis is still limited. COUP-TFII, a transcription factor expressed in venous but not arterial endothelial cells (ECs), has been shown to regulate venous identity via the inhibition of NOTCH mediated signals (You et al., 2005). Vice versa, NOTCH activation has been shown to down-regulate COUP-TFII expression (Swift et al., 2014). Furthermore, Akt activation was shown to inhibit Raf1-ERK1/2 signaling in ECs to favor venous specification (Ren et al., 2010). To date, however, the specific factors upstream of the Akt pathway that define venous EC identity remain unclear.
Tie2 is a receptor tyrosine kinase that mediates angiopoietin signaling for EC survival, vascular remodeling and integrity (Augustin et al., 2009). Tie2 deficiency led to embryonic lethality resulting from the defective vascular remodeling and heart development (Dumont et al., 1994; Sato et al., 1995), and combined deletion of Tie2 ligands Ang1 and Ang2 in mice was also shown to disrupt Schlemm’s canal formation leading to ocular hypertension and glaucoma (Thomson et al., 2014). Patients with venous malformations were shown to have Tie2 missense point mutations (Vikkula et al., 1996), leading to ligand-independent Tie2 activation (Limaye et al., 2009). However, the underlying mechanism of Tie2 function in the blood vessels has not been fully elucidated. In this study, we show that Tie2 deficiency or insufficiency induced by gene targeting leads to defective vein formation and maintenance during embryogenesis and postnatal development. Findings from this study suggest that Tie2 is essential for the specification of venous EC identity via the Akt mediated regulation of COUP-TFII protein stability.
To characterize Tie2 function in vascular development, a conditional knockout mouse model targeting the Tek gene (Shen et al., 2014a) was employed in this study. Ubiquitous deletion of Tek led to embryonic lethality by E10.5 (Figure 1—figure supplement 1A–C), as previously reported (Sato et al., 1995). As shown in Figure 1A,B and Figure 1—figure supplement 1D,E, no veins (arrows) were detected in the head or somite regions of the Tek null embryos at E9.5, unlike in the littermate control embryos. Interestingly, Tie2 expression in the E9.5 embryos was higher in veins than in arteries (arrowhead, Figure 1B). The lack of veins in the intersomitic regions of Tek deleted mice was also evident by Dll4 and PECAM-1 double staining (Figure 1—figure supplement 1E).
To investigate the role of Tie2 later during embryogenesis, we employed the Ubc-CreERT2 and Cdh5-CreERT2 deletor mouse lines (Wang et al., 2010), to generate the doubly transgenic mice (TekFlox/−/Ubc-CreERT2, named Tek−/iUCKO; or TekFlox/−/Cdh5-CreERT2, named Tek−/iECKO). TekFlox/+/Ubc-CreERT2 or TekFlox/+/Cdh5-CreERT2 littermate mice were used as controls. Tie2 deletion efficiency was examined by immunostaining with skin and also by real-time RT-PCR with lung of Tek mutant mice (Tek−/iECKO: 0.33 ± 0.07, n = 3; Tek+/iECKO: 1.0 ± 0.26, n = 3; Tek−/iUCKO: 0.25 ± 0.02, n = 4; Tek+/iUCKO: 1.0 ± 0.11, n = 4). Analysis of veins in skin at E15.5 showed that venogenesis was disrupted in both types of mutant embryos, in which Tek deletion was induced by intraperitoneal injections of tamoxifen into pregnant mice at E10.5–12.5 (Figure 1C–E and Figure 1—figure supplement 2A). In addition, defective vein formation was also observed in the skin of E17.5 Tek−/iUCKO embryos when Tek deletion was induced at E12.5–14.5 (Figure 1F). Lack of bleeding or edema with the later targeting suggests a stage-dependent function of Tie2 during the establishment of vascular integrity. Interestingly, veins were detected in mesentery, but unlike in littermate controls, they did not align properly with the arteries (Figure 1—figure supplement 2B). Furthermore, lymphatic vessels originate mainly from veins during embryonic development in mammals. It is therefore interesting to find out whether lymphatic development is altered in Tie2 knockout mice when the vein formation is defective. We found that lymphatic vessels were present in the skin of Tek−/iECKO mutant mice, but became dilated (Figure 1—figure supplement 3). This may be secondary to tissue edema resulting from the impairment of blood vasculature. However, it is also possible that Tie2 may have a role at earlier stages of lymphatic development.
To study the role of Tie2 in postnatal vascular formation, the TekFlox/−/Ubc-CreERT2 neonatal mice were treated with tamoxifen via intragastric injection during postnatal days (P1–4) and analyzed at P7 (Figure 2A). The efficiency of Tek deletion was examined by Western blot analysis, immunostaining and real-time RT-PCR. Little Tie2 protein was detected in the lung and retina of Tek−/iUCKO mice (Figure 2B,C). The level of Tie2 mRNA in the same tissues of Tek−/iUCKO mice was approximately 13–18% of the control mice as shown in Table 1. Consistent with the results from Western blot analysis, the deletion efficiency of Tie2 as analyzed by real-time RT-PCR in lungs of Tek−/iECKO mice was lower than that of Tek−/iUCKO mutants (Tek−/iECKO: 0.40 ± 0.10, n = 4; Tek+/iECKO: 1.0 ± 0.21, n = 4). The partial Tie2 deletion was also indicated by the mosaic GFP expression when the mTmG allele was crossed into the Tek mutant mice to generate the compound genetic mouse model (Tek−/iECKO;mTmG; Figure 2—figure supplement 1A–B) (Muzumdar et al., 2007). Interestingly, the decrease of Tie2 in the Tek−/iUCKO and Tek−/iECKO mice resulted in a significant decrease of retinal vascularization, as indicated by a lower vascular index (Figure 2C–F). As Tie2 is also expressed by some hematopoietic cells, we generated mutant mice with Tek deletion in blood cells (TekFlox/−;Vav-iCre) (de Boer et al., 2003). No obvious defects were observed in the retinal blood vessels of the mutant mice (Figure 2—figure supplement 2A,B).
In spite of the decreased vascular index in retina, there was a significant increase of vascular density at the front of the venous but not arterial segments of Tek−/iUCKO mutants compared with controls at P7 (Figure 2G–J). Biochemical analysis revealed a significantly decreased phosphorylation of the Dok-2 docking protein (pDok2 / beta-actin, Tek−/iUCKO: 0.38 ± 0.25, n = 6; Control: 1.0 ± 0.24, n = 6; p=0.0014; pDok2 / tDok2, Tek−/iUCKO: 0.52 ± 0.10, n = 3; Control: 1.0 ± 0.21, n = 3; p=0.024), but an increase of Erk1/2 phosphorylation (pERK1/2 / tERK1/2, Tek−/iUCKO: 1.71 ± 0.70, n = 10; Control: 1.0 ± 0.30, n = 9; p=0.012) in the lungs of Tek−/iUCKO mice compared with controls (Figure 2K,L). It is worth noting that the total Dok-2 protein level also decreased in the mutant mice after Tie2 reduction compared with that of the littermate control.
The scheme for Tek deletion by intragastric administration of tamoxifen was shown in Figure 3A. The abnormal angiogenesis along the retinal veins was also seen at P11 in mice with postnatal Tek deletion, and the morphology of veins was severely disrupted at P15 (Figure 3B and C, arrows). Vein degeneration was accompanied by the formation of haemangioma-like vascular tufts by P21 (Figure 3D, arrows). The massive angiogenic vascular growth occurred mainly in the first layer of retinal vessels of Tek−/iUCKO mice (Figure 3E). In contrast to wildtype littermate control mice, Tek−/iUCKOmutant mice at P21 exhibited little blood vessel growth towards the deep layers of retina (Figure 3E). Vessels in the vascular tufts in the Tek−/iUCKO mice had lumens and were covered with NG2+ pericytes (Figure 3E). Furthermore, Tek deletion at a later stage (P5-8) resulted in a similar but milder vascular phenotype (Figure 3F, arrows). For comparison, we also performed the analysis of retinal blood vasculature at different time points with Tek-/iECKO mice, but did not observe the formation of vascular tufts. This may be due to the lower efficiency of Tek gene deletion as discussed above.
Despite the lethality of Tek null embryos, the Tek−/iUCKO mice deleted at P1-4 survived with reduced body weight (Figure 3—figure supplement 1). Unlike in the retina, postnatal attenuation of Tie2 did not produce an obvious effect on blood vessels in tail skin at P7 (Figure 3—figure supplement 2A), or ear skin at P21 (Figure 3—figure supplement 2B). However, veins in ear skin were found to be tortuous in 2.5 month-old adult mutants compared with the control mice (Figure 4A, arrows). The expression analysis by real-time RT-PCR revealed that Tek transcript level remained low in Tek−/iUCKO mice at this stage (Tek−/iUCKO: 0.26 ± 0.14, n = 5; Control: 1.0 ± 0.28, n = 5). Interestingly, the venous tortuosity was also observed in retinas of Tek−/iUCKO mice at the adult stage (2.5 month-old) while the increased angiogenesis along the retinal veins regressed (Figure 4B). The findings suggest that Tie2 has an important role in the postnatal maintenance of veins.
Mechanistically, we found that Tie2 attenuation led to the alteration of venous EC identity as shown by the change of EC marker expression (Figure 5A, and Table 1). Transcript levels of the venous marker APJ and EphB4 were decreased, whereas COUP-TFII mRNA level was unaltered in the retinas and lungs of Tek−/iUCKO mice compared with littermate controls (P7; Figure 5A). Furthermore, there was a significant increase of arterial and angiogenic sprout marker Dll4 transcripts in the lungs (Figure 5A) (Hellström et al., 2007). Consistently, Tie2 reduction induced Dll4 expression in retinal veins (P9; Figure 5B, arrows point to veins in dotted regions), which were negative by immunostaining for Dll4 in littermate control mice (Figure 5C). There was no significant alteration of other arterial markers including NRP1, EphrinB2 and NOTCH1 (P7; Figure 5A). Furthermore, we also examined the expression level of some venous and arterial markers, including EphB4, APJ, Ephrin B2 and Dll4 in Tek mutant and control mice at the adult stage. Consistently, we found that venous genes were significantly decreased in lung of Tek−/iUCKO mice compared with controls (EphB4, Tek−/iUCKO: 0.52 ± 0.29, n = 5; Control: 1.0 ± 0.18, n = 5; APJ, Tek−/iUCKO: 0.22 ± 0.22, n = 5; Control: 1.0 ± 0.25, n = 5). Interestingly, there was also a trend of reduction in the arterial gene expression in Tek−/iUCKO mice (Ephrin B2, Tek−/iUCKO: 0.71 ± 0.32, n = 5; Control: 1.0 ± 0.23, n = 5; Dll4, Tek−/iUCKO: 0.70 ± 0.36, n = 5; Control: 1.0 ± 0.21, n = 5), suggesting that Tie2 retardation may also affect arteries at later stages.
Tie2 deletion led to a decrease of Akt phosphorylation (Ser473, Figure 6A,B). Interestingly, COUP-TFII protein was significantly decreased in lung and liver tissues of Tek mutant mice (Figure 6A,B and Figure 6—figure supplement 1A,B), although COUP-TFII mRNA level was not altered, as shown above. This was further verified in cultured HUVECs, where COUP-TFII protein was decreased when Tie2 was reduced by siRNA mediated knockdown (Figure 6C–D). Consistently, COUP-TFII protein was significantly increased by the COMP-Ang1 stimulation of Tie2/Akt pathway (Figure 6E–F). As the Tie2 downstream Akt activation was significantly suppressed in mice with Tie2 attenuation, we also analyzed COUP-TFII level when the PI3K/Akt pathway was blocked. COUP-TFII protein was significantly reduced 6 hr or 12 hr after treatment with the PI3K inhibitor LY294002 (Figure 6G,H; LY-3h: 1.11 ± 0.39, n = 5; LY-6h: 0.62 ± 0.12, n = 5; LY-12h: 0.39 ± 0.13, n = 5; values from five independent experiments normalized against control at three time points respectively). This was further confirmed with the Akt inhibitor MK2206, and there was a significant decrease of COUP-TFII after Akt inhibition at the 12 hr time point (Figure 6I,J; MK-3h: 1.32 ± 0.47, n = 7; MK-6h: 0.75 ± 0.31, n = 7; MK-12h: 0.23 ± 0.18, n = 7; values from seven independent experiments normalized against control at three time points respectively). Furthermore, the decrease of COUP-TFII protein by the Akt inhibition could be reverted by the treatment with a proteasome inhibitor MG132 (Figure 6K–L). These findings suggest that the Tie2 signaling pathway controls vein specification via Akt-mediated regulation of COUP-TFII protein stability (Figure 6M).
We show here that Tie2 is more expressed in veins than arteries in early embryos and in retina of neonate mice with the newly formed arteries expressing low level of Tie2. Interestingly, Tie2 is also downregulated in the sprouting tip endothelial cells as observed in retinal angiogenesis in this study and also by others (Augustin et al., 2009). It has been recently reported that vein-derived endothelial tip cells contribute to the emerging arteries during mouse retinal vascularization and also in zebrafish fin regeneration (Xu et al., 2014). This suggests that the downregulation of Tie2 may be required for the initial establishment of an arterial EC identity. Consistently, we have demonstrated in this study that Tie2 absence or insufficiency by gene targeting disrupts venogenesis during embryogenesis and postnatal development. At the molecular level, we have found that Tie2 participates in the determination of venous EC identity, which may act via Akt-mediated regulation of COUP-TFII protein stability. This implies that Tie2/Akt signaling counterbalances VEGFR2/MAPK pathway in arteriovenous specification during the vascular development. The findings are consistent with previous literature on the role of Akt in venous development, and with the recent report that cardiomyocyte derived Ang1 is required for the subepicardial coronary vein formation (Deng et al., 2013; Ren et al., 2010; Zimmermann and Moelling, 1999; Lamont and Childs, 2006; Arita et al., 2014).
As Tie2 is expressed by endothelial cells and non-endothelial cells such as hematopoietic cells, we employed three Cre deletors in this study to analyze the role of Tie2 in blood vascular development, including a EC-specific Cre line Cdh5-CreERT2, a hematopoietic Cre line Vav-iCre and a ubiquitous Cre line Ubc-CreERT2. Blood vessels developed normally in mice with Tek deletion in blood cells, while disruption of vein development was observed in Tek−/iUCKO as well as in Tek−/iECKO mutant mice. This suggests that the disruption of cutaneous vein development during embryogenesis is likely to be a cell-autonomous effect resulting from the deletion of Tie2 in endothelial cells. In postnatal studies, we found that there was a dramatic increase of blood vessel growth after the ubiquitous Tie2 attenuation, which led to the formation of haemangioma-like vascular tufts along the retinal veins in the Tek mutant mice. Although a significant decrease of retinal vascularization was observed in Tek−/iECKO mice, we did not observe the vascular tuft formation in the retina of these mutants. This may result from the lower Tie2 deletion efficiency in Tek−/iECKO mice when compared with that of Tek−/iUCKO mice. However, we cannot completely rule out the possibility that Tie2 deletion in other non-endothelial cells may contribute to the vascular abnormalities in retinas of Tek−/iUCKO mutant mice. As retinal neovascularization is one of the frequent causes of vision loss in patients with proliferative diabetic retinopathy and neovascular age-related macular degeneration (Gariano and Gardner, 2005), these data suggests that reduction of Tie2 mediated signals in ECs and / or other Tie2 expressing cells may be implicated in the vascular pathologies.
COUP-TFII is expressed by several cell types including endothelial cells (Wu et al., 2016). In the vascular system, COUP-TFII has been shown to regulate venous EC identity as well as lymphatic EC specification (You et al., 2005; Lin et al., 2010; Srinivasan et al., 2010). At the transcription level, COUP-TFII is regulated by several factors / pathways including NOTCH and SOX7/18 (Swift et al., 2014). In this study, we have found that COUP-TFII protein but not its mRNA level is significantly reduced in genetically engineered mouse model targeting Tek. Based on the evidence obtained from the biochemical analysis with cultured endothelial cells, we propose that Akt is a strong candidate mediating Tie2 signals in this process. As Akt acts downstream of many signaling pathways and that it is also activated in many cell types, it remains to be investigated how Akt regulates COUP-TFII protein stability in venous ECs. Also, further in vivo mechanistic studies are required for validating and fully elucidating Akt and others factors / pathways downstream of Tie2 in venous specification. In addition, we have also found that the requirement of Tie2 for vein development differs between the skin and mesentery. When Tie2 levels were reduced by gene targeting, veins were mostly undetectable in the skin, but displayed only abnormal arterial-venous alignment in the mesentery. It is possible that the cutaneous veins are more sensitive to the loss of Tie2 mediated signals, or that venogenesis occurs earlier in the mesentery than in the skin. The abnormal arteriovenous alignment resembled that of the APJ deficient mice (Kidoya et al., 2015), and a significant reduction of APJ in the Tek mutants suggests that APJ acts downstream of Tie2 in the regulation of venous patterning.
Interestingly, we have also found that both the total and phosphorylated Dok-2 decreased significantly after Tie2 attenuation in this study. As Dok family members have been shown to negatively regulate ERK1/2 activation (Honma et al., 2006), the decrease of Dok-2 may lead to the increase of retinal angiogenesis as detected in mice with Tie2 reduction by gene targeting. Consistently, enhancement of Tie2 signaling via the inhibition of VE-PTP has been shown to stabilize blood vessels and suppress retinal angiogenesis in mouse models of ischemia-induced retinal neovascularization (Shen et al., 2014b). Furthermore, in experiments using HEK 293 T or endothelial cells overexpressing Dok2, it was found that Dok2, via directly interacting with Tie2, mediated Tie2 pathway in endothelial cell migration (Saharinen et al., 2008; Master et al., 2001). Decreased Dok-2 activation in Tek mutants may also account for the suppression of horizontal and vertical blood vessel growth during retinal vascular network formation. So far, in vivo evidence about the role of Dok2 in blood vascular development is still missing. Further studies employing animal models targeting Dok2 are important to analyze its biological functions in the vascular system.
In summary, we show that Tie2 is required for the specification and maintenance of venous EC identity. Related to the findings of this study, activating mutations of Tie2 have been linked to venous malformations in patients (Vikkula et al., 1996). It is still poorly understood about the difference between the pathways downstream of activating Tie2 mutants and the wildtype Tie2. Activation of wildtype Tie2 by its ligand Ang1 or VE-PTP inhibition, at least for a short period, has been shown to stabilize blood vasculature (Shen et al., 2014b), while activating Tie2 mutations lead to venous malformations (Vikkula et al., 1996; Limaye et al., 2009; Boscolo et al., 2015). Mechanisms underlying the discrepancy require further investigation. As alteration of Tie2 mediated signals may be implicated in a variety of vascular pathologies associated with the venous system, further investigation along these lines may help to develop novel Tie2-targeted therapeutics.
Conditional mice with Tek gene targeted flox sites (TekFlox) for gene deletion were generated by the National Resource Center for Mutant Mice, Nanjing University, as previously described (Shen et al., 2014a). All animal experiments were performed in accordance with the institutional guidelines of the Soochow and Nanjing University Animal Center (MARC-AP#YH2). To generate mice with ubiquitous or cell-specific Tek gene deletion, TekFlox mice were crossed with transgenic mice expressing Cre recombinanse in ECs (Cdh5-CreERT2) (Wang et al., 2010), hematopoietic cells (Vav1-iCre) (de Boer et al., 2003), or ubiquitously (Ubc-CreERT2 or EIIa-Cre) (Lakso et al., 1996; Ruzankina et al., 2007). The floxed mice used in this study were maintained in C57BL/6J (RRID:IMSR_JAX:000664) with at least five backcrosses. In all the phenotype analysis, littermates were used as control.
Induction of gene deletion was performed by tamoxifen treatment as previously described (Shen et al., 2014a). Briefly, pregnant mice were treated with tamoxifen (Sigma-Aldrich) at E10.5–12.5 or E12.5–14.5 (1–2 mg/per mouse for three consecutive days by intraperitoneal injection), and analyzed later. New-born pups were treated with tamoxifen by four daily intragastric injections and after P7 by four daily intraperitoneal injections, and analyzed later.
Tissues from Tek mutant and control mice were collected and homogenized in TRIzol (Ambion). RNA extraction and reverse transcription were performed by standard procedures (RevertAid First Strand cDNA Synthesis Kit, Thermo Scientific). Quantitative real-time RT–PCR was carried out using the SYBR premix Ex Taq kit (TaKaRa). Briefly, for each reaction, 50 ng of total RNA was transcribed for 2 min at 50°C with a denaturing step at 95°C for 30 s followed by 40 cycles of 5 s at 95°C and 34 s at 60°C. Fluorescence signal was analyzed by using ABI PRISM 7500. The primers used were as follows: GAPDH: 5'-GGTGAAGGTCGGTGTGAACG-3', 5'-CTCGCTCCTGGAAGATGGTG-3'; Tie2: 5'-GATTTTGGATTGTCCCGAGGTCAAG-3', 5'-CACCAATATCTGGGCAAATGATGG-3'; APJ: 5’-CAGTCTGAATGCGACTACGC-3', 5'-CCATGACAGGCACAGCTAGA-3'; Ephb4: 5'-CTGGATGGAGAACCCCTACA-3', 5'-CCAGGTAGAAGCCAGCTTTG-3'; COUP-TFII: 5'-GCAAGTGGAGAAGCTCAAGG-3', 5'-TTCCAAAGCACACTGGGACT-3'; NRP1: 5’-CCGGAACCCTACCAGAGAAT-3', 5'-AAGGTGCAATCTTCCCACAG-3'; EphrinB2: 5'-TGTTGGGGACTTTTGATGGT-3', 5'-GTCCACTTTGGGGCAAATAA-3'; NOTCH1: 5'-TGTTGTGCTCCTGAAGAACG-3', 5'-TCCATGTGATCCGTGATGTC-3'; Dll4: 5'-TGCCTGGGAAGTATCCTCAC-3', 5'-GTGGCAATCACACACTCGTT-3'. The transcripts of venous and arterial markers were normalized against GAPDH, and the relative expression level of every gene in the Tek mutants (Tek−/iUCKO) was normalized against that of littermate control mice.
To analyze Tie2 mediated signaling pathway, lung tissues from Tek mutant and control mice were homogenized following standard procedures. Antibodies used included rabbit polyclonal anti-Tie2 (Santa Cruz sc-324, RRID:AB_631102), rabbit polyclonal anti-Akt (Cell Signaling Technology #9272, RRID:AB_329827), rabbit monoclonal anti-phospho-Akt473 (Cell Signaling Technology, #4060), rabbit polyclonal anti-phospho-p56Dok-2 (Cell Signaling Technology #3911, RRID:AB_2095082), rabbit polyclonal anti-p56Dok-2 (Cell Signaling Technology #3914, RRID:AB_2095080), rabbit polyclonal anti-Erk1/2 and phospho-Erk1/2 (Cell Signaling Technology #9101, RRID:AB_331646; and 9102, AB_330744), mouse monoclonal anti-COUP-TFII (R and D Systems #PP-H7147-00, RRID:AB_2155627), and mouse monoclonal to beta–actin antibody (C4, Santa Cruz sc-47778, RRID:AB_626632).
HUVECs (human umbilical vein endothelial cell, C0035C, GIBCO) were cultured in endothelial cell basal medium plus supplements (#M200-500 GIBCO, or #1001 ScienCell Research Laboratories). To knock down Tie2 expressoin in HUVECs, cells were transfected with Stealth RNAiTM siRNA duplex oligoribonucleotides targeting human Tie2 (HSS110623, HSS110624 and HSS110625; Invitrogen) using Lipofectamine RNAiMax (Invitrogen, CA, USA); Stealth RNAi negative control duplexes (medium GC) were used as a control. COMP-Ang1 (kind gift from Dr. Gou Young Koh; 0.2–0.4 μg/ml) or Ang1 (923-AN, R and D) was used to stimulate Tie2/Akt pathway. To inhibit PI3K and downstream Akt signaling, HUVECs were treated with LY294002 (40 μM, S1105, Selleckchem) or MK2206 (10 μM, S1078, Selleckchem) and analyzed at 3 hr, 6 hr or 12 hr after treatment. For the experiment with proteasome inhibition, HUVECs were treated with MG132 (10 μM, s2619, Selleckchem). The cells were washed with ice-cold PBS and lysed in the lysis buffer (1 mM PMSF, 2 mM Na3VO4, 1× protease and phosphatase inhibitor cocktail without EDTA (Roche Applied Science), 20 mM Tris-HCl pH 8.0, 100 mM NaCl, 10% glycerol, 50 mM NaF, 10 mM β-glycerolphosphate, 5 mM sodium pyrophosphate, 5 mM EDTA, 0.5 mM EGTA and 1% NP-40). The lysates were incubated on ice for 0.5 hr with rotation and centrifuged. Protein concentration was determined using the BCA protein assay kit (PIERCE), and equal amounts of protein were used for analysis.
For whole-mount immunostaining with embryonic skin, mesentery and retina, tissues were harvested and processed as previously described (Shen et al., 2014a). The tissues were fixed in 4% paraformaldehyde, blocked with 3% (w/v) milk in PBS-TX (0.3% Triton X-100), and incubated with primary antibodies overnight at 4°C. The antibodies used were rat anti-mouse PECAM-1 (BD Pharmigen, 553370, RRID:AB_394816), goat anti-mouse Tie2 (R and D, AF762, RRID:AB_2203220), goat anti-mouse Dll4 (R and D, AF1389, RRID:AB_354770), goat anti-mouse EphB4 (R and D, AF446, RRID:AB_2100105), Cy3-conjugated mouse anti-mouse αSMA (Sigma, C6198, RRID:AB_476856). Alexa488, Alexa594 (Invitrogen), Cy5- or Cy3- (Jackson) conjugated secondary antibodies were used for staining. Slides were mounted with Vectashield (VectorLabs), and analyzed with the Olympus FluoView 1000 confocal microscope or Olympus BX51 fluorescent dissection microscope. For staining of frozen sections, retinas were collected and fixed in 4% PFA for 1 hr at 4°C, incubated in 20% sucrose overnight and then embedded in OCT. Consecutive sections (10 µm in thickness) were incubated with antibodies against PECAM-1 (BD Pharmingen, 553370, RRID:AB_394816) and NG2 (Millipore, AB5320, RRID:AB_11213678), followed by staining with the appropriate fluorochrome-conjugated secondary antibodies and mounted as described above.
Retinal vascularization index was quantified as the ratio of vascularized area to total retinal area. For the quantification of blood vessel parameters in the retina, fluorescent images were taken from similar regions in all samples. Blood vessel density was measured and analyzed by using Image Pro Plus (MediaCybernetics), as previously described (Shen et al., 2014a).
Statistical analysis was performed with the unpaired t test. All statistical tests were two-sided. Data are presented as mean ± S.D.
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Elisabetta DejanaReviewing Editor; FIRC Institute of Molecular Oncology, Italy
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Thank you for submitting your work entitled "Angiopoietin receptor Tie2 is required for vein specification and maintenance via regulating COUP-TFII" for consideration by eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Janet Rossant as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Hellmut Augustin (Reviewer #2); Lena Claesson-Welsh (Reviewer #3).
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All the reviewers agree that the paper contains interesting and rather novel observations. However they feel that the paper needs additional experiments to make the results stronger and to get a more mechanistic view of the effect of Tie2 in venous differentiation. Both reviewers 2 and 3 list major points to be considered and specific experiments to add to improve the manuscript. Since these revisions require considerable additional work, we are not able to consider a revised manuscript.
We encourage the authors to complete the recommended revision of their work and consider resubmitting to eLife in the future.
The paper by Man Chu et al. describes the effect of inactivation of Tie-2 expression on artero-venous differentiation in the mouse embryo, pups and cultured endothelial cells.
As previously reported, Tie 2 null embryos die early during gestation for vascular and heart developmental problems. Here the authors show that Tie-2 deficient embryos and pups present a defective venous organization when Tie-2 expression is missing. In some organs and tissues, veins are absent along the arteries. In the retina there is a reduced horizontal growth of the vasculature and the presence of malformations along the length of the veins. In the skin, veins look tortuous. Arterial and venous markers are partially modified. In HUVEC silenced for Tie2 or lung and liver tissues Coup-TFII is reduced.
Mechanistically, data suggest that Tie-2 may act through phosphorylation of Akt that in turn would increase COUP-TFII and venous differentiation.
Although the paper contains some interesting observations it is essentially descriptive. The major weakness is the lack of a detailed mechanistic analysis of how Tie-2 acts in inducing venous differentiation. Data on the role of Akt are suggestive but a marked reduction of COUP-TFII is detectable only in HUVEC KD for Tie-2 and is much less apparent in the tissues. Other published studies showed an increase in cultured cells of Akt and STAT 1 upon infection with Tie-2 gain of function mutant. In addition, inherited gain of function mutations of Tie-2 induce a strong venous endothelial cell proliferation and formation of large hemangiomas inhibited by Rapamycin (for instance Boscolo et al. J Clin invest). To my view the authors should extend their studies on the mechanism of action of Tie-2 also adding novel observations as compared to previously published literature.
The authors of the manuscript, "Angiopoietin receptor Tie2 is required for vein specification and maintenance via regulating COUP-TFII" have used global and endothelial cell-specific conditional Tie2 knockout mouse models to demonstrate the critical role of Tie2 during vein development. The study is novel and addresses a fundamental question regarding the pathways (Tie2, Akt/PI3K and COUP-TFII) involved in forming venous endothelial identity and function. Although there is already a lot of circumstantial evidence in the published literature on vein-specific functions of Ang/Tie signaling, the manuscript makes an important point in showing this conclusively using definite genetic models. The study will be of interest for the broad readership of the journal. In further advancing their work, the authors should consider the following:
1) The major limitation in the study is the use of a ubiquitously expressed Cre deletor line (UBC-CreERT2) to knockout Tie2. Even though the authors have also used an endothelial specific Cre line (Cad5-CreERT2) to demonstrate the phenotypes in embryos and early postnatal retina, all the mechanistic studies are only performed using the UBC-CreERT2 line. This is a major concern especially because Tie2-/iUCKO seems to show better Tie2 deletion as well as stronger phenotypes compared to Tie2-/iECKO line. Is this solely due to different recombination efficacies of the different drivers or would this difference raise the question whether endothelial cell autonomous effects may not be the only cause of defective vein development in Tie2-deficient mice? Why did the authors opt to study different time points in Figure 1D and E?
2) The immunofluorescence images show essentially black boxes in Figures 1A, B, D, E and in Figure 2B and C suggesting 100% deletion efficacy. Do the authors claim to have achieved 100% deletion of Tie2 in these experiments or why are these images not showing the expected mosaic staining pattern resulting from partial recombination? A quantitative analysis of the efficacy of Tie2 deletion in the different experiments should/needs to be included. Alternatively, qRT-PCR to show Tie2 expression should be done with retina and embryo tissues from Tie2-/iUCKO (tissues and isolated endothelial cells). In Figure 2A, the WB shows about 30-50% Tie2 in the Tie2-/iECKO mice which is clearly not corresponding to the extremely weak or absent Tie2 staining shown in Figure 2C.
3) The retina vasculature analysis at P11, P15 and P21 in Tie2-/iUCKO mice showing vascular tuft formation is interesting. The same analysis should be included using the Tie2-/iECKO mice.
4) Although hemangioma-like vascular tufts were observed in Tie2-/iUCKO mice at P21, the adult retina did not show any such malformations, whereas ear skin showed tortuous veins in adult mice. These data have been discussed rather insufficiently. The authors should comparatively analyze COUP-TFII transcript levels and other downstream players such as pAkt and pDok-2 in these two vascular beds in order to dissect the mechanisms, which help the retinal veins to "recover" but not the cutaneous veins. Likewise, partial recombination will lead to a mosaic vasculature of WT and KO cells. If such mosaic mice are traced over time, there is a good chance that the KO cells will be competed out by WT cells. As such, it is critical, particularly in the longer term experiments to carefully trace the percentage of cells with Tie2 deletion at the later time points of analysis.
5) The authors claim that Tie2 is required for the maintenance of venous EC identity. This is suggested by the observed phenotypes (albeit only moderately convincing). A systematic gene expression analysis of adult retina similar to Figure 5A (performed using P7 retina and lung) is missing. It is important to know whether the expression of venous markers is restored over time and also whether Tie2 expression is recovered in the adult mice as a result of incomplete Cre recombination efficiency and compensatory proliferation of Tie2-expressing EC (see above).
6) The authors are encouraged to reconsider their discussion. They conclude at the end of the discussion that the findings put a cautionary note on long term Tie2 targeting therapies, e.g., during tumor angiogenesis. This may not be the most important point of the study. In fact, nobody tries to target Tie2. Ang2 neutralizing strategies primarily work cooperatively with VEGF pathway targeting drugs based on their vascular normalization Tie2 gain-of-function effect. The same holds true for VE-PTP inhibitors or the recently published ABTAA antibody. What this reviewer finds much more exciting is the role of Tie2 on venogenesis. Venogenesis has mostly been considered as the default pathway with transcriptional programs driving lymphatic and arterial differentiation. The identification of Tie2 signaling as upstream regulator of COUP-TFII is a major finding that should likely be discussed in more detail. The fact that Tie2 KO leads in part to the acquisition of an arterial phenotype is noteworthy and suggests something of an arteriovenous identity balance mechanism. Likewise, angiogenesis is an arterializing process. As such, the physiological downregulation of Tie2 in the angiogenic tip cell vasculature should/could likely be discussed in the context of the findings of this manuscript. Obviously, it is at the discretion of the authors to prioritize the discussion, but it is felt that the authors may in the present version of the manuscript not have discussed the most obvious and most exciting implications of their work.
The study by Chu et al. characterizes the effect of Tie2 gene inactivation either using a ubiquitous or endothelial promoter to drive Cre expression during different stages of embryonic and postnatal stages. Interestingly, Tie2 deficiency leads to loss in vein formation or in stage-dependent maintenance of veins in different organs. The analyses are thorough and neatly presented. The study is ambitious, novel and of general interest.
1) The study shows that loss of Tie2 is accompanied by vascular abnormalities and a deficiency in vein formation. It is not entirely clear from the images that veins are missing entirely or if they are malformed. Please complement images with aSMA stainings (which should outline the arterial tree).
2) The author have previously described the iUCKO-/- mice in their paper in ATVB Jun;34(6):1221-30. Here, they have examined lymphatic vessel formation in the skin and show that deletion of Tie2 in neonate mice did not affect lymphatic vessel growth and maturation (Figure 8). I'm surprised that the lack of veins, as shown in the current study, has had no consequence for lymphatic vessel formation. The authors need to clarify and confirm that lymphatics are unaffected although veins are lacking.
3) In Figure 1D, the authors examine embryos at E15.5 and in panel E, at E17.5. However, the embryos shown in the E panel seem considerably younger than the ones in the D panel? Magnification bars are missing for the embryos
4) In Figure 5, the authors show no difference in transcript levels of COUP-TFII when comparing retina and lung tissues from WT and iUCKO-/- mice. When analyzing protein levels (Figure 6) in lung tissue from mutant mice vs WT, the differences are now significant. Does loss of Tie2 lead to increased turnover of COUP-TFII? As COUP-TFII is a critical regulator of vein and lymphatic development, it is important to learn more about how it's regulated by Tie2. This has a bearing also on the (lack of?) effect of Tie2 deletion on lymphatic development.
[Editors’ note: what now follows is the decision letter after the authors submitted for further consideration.]
Thank you for submitting your work entitled "Angiopoietin receptor Tie2 is required for vein specification and maintenance via regulating COUP-TFII" for consideration by eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by a Senior Editor.
While the reviewers feel that your work is potentially very important, they were disappointed that you chose to argue points rather than making the necessary revisions elaborated in the original set of reviews. You will see from their comments, that none of the reviewers felt you adequately addressed their original concerns. Normally, eLife only allows for a single round of reviews. Given the importance of your work and the fact that all three reviewers have urged me to give you another opportunity to revise the paper, we are willing to give you a second chance to make the original required major revisions. I would like to stress that the reviewers are your advocates and trying to help you improve the paper. The reviewers’ comments from the second round of review are listed below, but I urge you to make the revisions requested in both the first and second round of review.
This will be your final opportunity to provide acceptable additional experimental evidence as required by the reviewers.
Overall my feeling is that the authors could have a very nice story but they do not want to make an effort to make it more complete. Data on the mechanism of action remain the same, as poor as before.
I do not think that they can conclude that since COUP-TFII is reduced when Akt activation is inhibited (in HUVEC) Akt activation is the mechanism of action of Tie-2 in maintaining a venous identity. Although there is a partial inhibition of Akt (24% reduction) in Tie-2 deficient mice other pathways are likely implicated.
pDok- 2, for instance, seems more dramatically affected. It is sad that they do not want to make a relatively small effort to improve their paper.
The same applies to the relevant points raised such as the use of a model of ubiquitous inactivation of Tie-2 versus endothelial specific inactivation, or the lack of studies on the possible alterations of the lymphatics in absence of a correct venous development.
The manuscript by Man Chu and coworkers is resubmitted after revision. That global deletion of Tie2 casus venous defects is convincingly demonstrated. I have the following comments:
1) A major concern with this study is the weak phenotype of the endothelial deletion compared to the ubiquitous deletion. The strong effects in the global deletion could be primary as well as secondary.
2) The question on how lack of veins affect lymphatics remains unanswered. The authors published in ATVB in 2014, that lymphatic vessel development is unaffected in neonate pups with the same global Tie2 deletion as described here. It is unexpected that the lack of veins would not affect lymphatic development.
3) Mechanistic in vivo data remain scarce.
This reviewer is rather disappointed on how the authors have addressed the specific critique to the original submission of the manuscript. The idea of the peer review process is to initiate an iterative process aimed at improving the eventually published manuscript. As such, this reviewer had constructively raised a number of issues that he felt would help to improve the manuscript. Generally speaking, the authors have not accepted this invitation, but essentially argued away all of the reviewer's critique and suggestions. They thereby missed the opportunity to improve their manuscript (at least in response to the comments and suggestions made by this reviewer). For example, they did not even opt to replace the black IF images for the supposed Tie2 ECKO analyses. In the rebuttal, they argue at length about different recombination efficacies using the two different driver lines. Clearly, incomplete recombination and the resulting competition of WT and KO cells in long term experiments is key to the interpretation of conditional KO data. If the authors' recombination analyses are correct, then this reviewer would insist that the corresponding IF images should show mosaic Tie2 expression and not black images.
Despite this disappointment about the revised manuscript, this reviewer considers the manuscript of high quality and in principle deserving of publication. As such, if the authors are either not able or not willing to adequately address this reviewer's concern and suggestions, the manuscript should be published as is.https://doi.org/10.7554/eLife.21032.017
- Yulong He
- Yulong He
- Yulong He
- Yulong He
- Yulong He
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
We thank Dr Dietmar Vestweber and Martina Dierkes for the discussion and kind help with experiments using COMP-Ang1, and staff in the Animal facility of Soochow University and Model Animal Research Institute of Nanjing University for technical assistance. This work was supported by grants from the National Natural Science Foundation of China (91539101, 31271530, 31071263), the Ministry of Science and Technology of China (2012CB947600), and the Priority Academic Program Development of Jiangsu Higher Education Institutions.
Animal experimentation: Conditional mice with Tek gene targeted flox sites for gene deletion were generated by the National Resource Center for Mutant Mice, Nanjing University. All animal experiments were performed in accordance with the institutional guidelines of the Soochow and Nanjing University Animal Center (MARC-AP#YH2).
- Elisabetta Dejana, Reviewing Editor, FIRC Institute of Molecular Oncology, Italy
© 2016, Chu et al.
This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.