Solar Flood Lights 100W Lumens
Outdoor 60W solar light manly use as a security Light with a Motion Sensor. Wireless, Waterproof design can be widely used to secure your outdoor space with added security and illumination. Turn on automatically when the sensor detects motion nearby within a wide range of 15 feet and 120 degrees angle. Further, it can work 12 hours with 100% lighting capacity or there is an internal motion sensor mode that can be selected by the given remote controller.Fluorescent lamp, would a 3' T12 Lamp produce more light than a 3' T8 Tube?t-8 32w produces 2600 lumens t-12 40w produces2520 lumens you wont be able to see the difference...t-8 would be my selection slightly more lumens less wattage.VE-PTP regulates VEGFR2 activity in stalk cells to establish endothelial cell polarity and lumen formationWe analysed sprouting angiogenesis in mouse embryoid bodies (EBs) from wild-type (WT) and ve-ptp/ embryonic stem cells (ESCs)8,21. Ve-ptp/ EBs formed a denser network of vessel sprouts with similar length but with increased area compared with WT EBs (Fig. 1a-c). There was a tenfold increase in CD31/VE-cadherin double-positive ECs in VEGF-treated ve-ptp/ EBs compared with VEGF-treated wild-type EBs (Fig. 1d). Moreover, the ve-ptp/ ECs extended numerous long filopodia throughout the sprout, while most WT stalk cells did not (Fig. 1e). We hypothesized that increased EC proliferation and filopodia formation might be due to elevated VEGFR2 activity. Indeed, immunostaining for VEGFR2 and the VEGFR2 phosphorylation site pY1175 (Fig. 1f) showed increased levels in ve-ptp/ sprouts compared with WT (Fig. 1g). The pVEGFR2/total VEGFR2 ratio was significantly higher in the ve-ptp/ stalks compared with WT stalks. The pVEGFR2 activity often colocalized with CD31 immunostaining, which was used to identify EC junctions (Fig. 1h). The VEGFR2 and pVEGFR2 stainings did not always colocalize, possibly because the antibodies against pVEGFR2 and VEGFR2 detected receptor intra- and extracellular domains, respectively. Immunostaining for VE-PTP also showed junctional localization (Fig. 1i). Supplementary Figure S1a,b shows that VE-PTP ablation was accompanied by reduced pericyte coating, indicating immature sprouts. Moreover, whereas ve-ptp transcripts were efficiently eliminated after gene targeting, there was no change in expression levels of genes known to affect angiogenic sprouting, such as delta-like 4, notch 1 and vegfr2 (Supplementary Fig. S1c-f). Substrates for VE-PTP include Tie2, an angiopoietin receptor implicated in control of vascular quiescence13. Lack of Ang1 or Tie2 leads to disturbed vascular remodelling during mouse embryonic development22,23,24. To compare VE-PTP's effect on VEGFR2 and Tie2, we employed a substrate-trapping, phosphatase-dead mutant of VE-PTP (D/A VE-PTP; aspartic acid 1180 in the catalytic domain exchanged for alanine). Substrate-trapping mutants bind their substrates without dephosphorylation25. Accordingly, expression of D/A VE-PTP allowed co-immunoprecipitation of pY992Tie2 with VE-PTP in Ang1-treated cells (Fig. 2a). Tie2 was co-immunoprecipiated also with WT VE-PTP, which was enzymatically active towards a standard substrate, Src optimal peptide (Supplementary Fig. S2a). The pY992Tie2 signal in response to Ang1 was weaker in cells expressing WT VE-PTP than D/A VE-PTP, indicating dephosphorylation of phosphorylated Tie2 by WT VE-PTP (Fig. 2a). Tie2 was also dephosphorylated in vitro using a purified VE-PTP catalytic domain fragment (Supplementary Fig. S2b). In contrast, expression of WT and D/A VE-PTP in VEGFR2-expressing Porcine aortic endothelial (PAE) cells lacking Tie2 expression did not allow co-immunoprecipitation of VEGFR2 with VE-PTP (Fig. 2b). The level of VEGFR2 phosphorylation remained unaffected by VE-PTP, in accordance with previous data20,26. However, immunoprecipitated pVEGFR2 was efficiently dephosphorylated in vitro, by the purified VE-PTP catalytic fragment (Supplementary Fig. S2b). Importantly, introduction of Tie2 together with VE-PTP and VEGFR2 in the PAE cells, followed by treatment with VEGFAng1, allowed VE-PTP-mediated pVEGFR2 dephosphorylation (Fig. 2c; Supplementary Fig. S3a,b). VEGFR2 moreover formed a complex with the D/A VE-PTP mutant in the presence of Tie2, in particular, in response to the combination of ligands (Fig. 2d). The protection by D/A VE-PTP of VEGFR2 from dephosphorylation was poor, possibly indicating that VEGFR2 was co-precipitated indirectly, via a D/A VE-PTP/Tie2 complex and therefore exposed to the action of other cellular phosphatases. The requirement for Tie2 in VE-PTP dependent dephosphorylation of VEGFR2 was further demonstrated by silencing either VE-PTP or Tie2 in primary ECs using short interfering RNA (siRNA). Thus, i) silencing of VE-PTP rendered cells insensitive to Ang1's effect on pVEGFR2 dephosphorylation (Fig. 2e) and ii) silencing of Tie2 increased VEGF-induced pVEGFR2 levels compared with the control (Fig. 2f). Combined, these results indicate that VE-PTP dephosphorylates VEGFR2 in a Tie2-dependent manner. Unlike the effect of silencing Tie2, introduction of Tie1 siRNA did not increase VEGF-induced accumulation of pVEGFR2, instead, there was a further decrease in pVEGFR2 levels in the Tie1-silenced cells (Fig. 2g). These data are in accordance with a negatively regulatory role for Tie1 visavi Tie2 (ref. 27). Furthermore, VEGFC-induced VEGFR3 tyrosine phosphorylation was not influenced by co-expression of Tie2 (Fig. 2h). A Tie2-truncated mutant, retaining the transmembrane domain but lacking the intracellular part including the kinase domain, partially decreased VEGFR2 phosphorylation in an Ang1-insensitive manner (Supplementary Fig. S3a). Introduction of a kinase-dead (KD) Tie2 mutant22 suppressed VEGFR2 phosphorylation to an extent similar to that seen for Ang1-treated cells expressing WT Tie2. The additional slight effect of Ang1 on cells expressing KD Tie2 seen here may reflect Tie2-independent effects of Ang1 (ref. 28). We conclude that Tie2 kinase activity is not required for full VE-PTP activity towards VEGFR2. We hypothesized that VE-PTP's effect towards Tie2 and VEGFR2 may be through formation of trimeric, constitutive complexes. To investigate a potential trimeric complex, we introduced V5-tagged VE-PTP and FLAG-tagged Tie2 into PAE/VEGFR2 cells that were treated or not with VEGF and Ang1, and performed consecutive immunoprecipitation and immunoblotting. The expression levels of VEGFR2, Tie2 and VE-PTP were comparable in the different conditions (Fig. 2i, left panel). Immunoprecipitation of Tie2 from cells expressing all three molecules followed by re-immunoprecipitation of VE-PTP allowed visualization of Tie2, VE-PTP and VEGFR2 in subsequent immunoblotting (Fig. 2i middle panel), showing the existence of a trimeric complex. We noted that the intracellular, lower molecular weight form of VEGFR2 was included in the complex. Immunoprecipitation of VEGFR2 from the supernatant of the anti-VE-PTP re-immunoprecipitation (that is, VE-PTP-depleted), showed co-immunoprecipitation of Tie2 with VEGFR2. We conclude that VEGFR2 existed both in a constitutive complex with Tie2 alone as well as in a trimeric complex with Tie2 and VE-PTP. Furthermore, the degree of complex formation was unaffected by VEGF and Ang1. VEGFR2 has been shown to colocalize and engage in complex with VE-cadherin29,30,31. 14). We asked whether VEGFR2 and Tie2 colocalized at cell-cell junctions. As seen in Fig. 3a, VEGFR2 and Tie2 translocated to cell-cell junctions in response to their respective ligand, as well as the combination of ligands (quantified in Fig. 3b). Indeed, VEGFR2 phosphorylation in VEGFAng1-treated, confluent ECs was reduced compared with treatment with VEGF alone. In sparse ECs, VEGF and VEGFAng1 induced similar levels of pVEGFR2 (Fig. 3c). In agreement, immunostaining for pVEGFR2 and VEGFR2 in confluent, VEGF-treated primary ECs showed pVEGFR2 localized to junctions after 5 and 60 min (Supplementary Fig. S4). In the presence of VEGFAng1, junctional pVEGFR2 levels were suppressed. These results strongly suggest that VE-PTP dephosphorylates VEGFR2 via Tie2 at cell-cell junctions. To further identify the subcellular localization of VE-PTP-regulated VEGFR2, we performed in situ proximity ligation assays (PLA) using antibodies against pVEGFR2 and VEGFR2 and oligonucleotide-ligated secondary antibodies. PLA products representing phosphorylated VEGFR2 increased with VEGF and decreased with VEGFAng1 treatment (quantification in Fig. 4b). Silencing of ve-ptp augmented PLA-detection of pVEGFR2 in response to VEGF; importantly, inclusion of Ang1 was without effect in VE-PTP-deficient cells. The pVEGFR2/VEGFR2 PLA spots were localized preferentially at or close to junctions in ve-ptp-silenced cells (Fig. 4a-c). Quantification of junctional localization showed fourfold induction of pVEGFR2/VEGFR2 in WT cells, compared with tenfold induction in ve-ptp-silenced cells. Controls for the PLA reactions (Supplementary Fig. S5) by omitting primary antibodies showed a high degree of specificity. We also used PLA to show that VEGFR2/VE-PTP complexes were abundant in the basal condition and decreased with VEGF, as reported previously20 (Fig. 4d). In response to VEGFAng1, the number of VEGFR2/VE-PTP complexes remained intact at the basal level when analysing the number of complexes per cell (Fig. 4e). However, the number of VEGFR2/VE-PTP complexes at cell-cell junctions increased with VEGFAng1 (Fig. 4f). Furthermore, in cells silenced for tie2, VEGFR2/VE-PTP complex formation was reduced and insensitive to VEGF (Fig. 4g). These data strengthen the notions that Tie2 modulates VE-PTP's interaction with VEGFR2 and that the three components communicate to balance quiescence preferentially at cell-cell junctions. We observed a reduced number of pericytes associated with angiogenic sprouts in ve-ptp/ EBs (Supplementary Fig. S1a,b); a hallmark of vessel immaturity. We therefore asked whether excess VEGFR2 activity in the absence of VE-PTP would interfere with vessel maturation. In WT sprouts at day 14 of differentiation, the apical/lumen markers podocalyxin and moesin32,33 were detected at stalk EC junctions, which contained a clearly distinguishable lumen (Fig. 5a, left). In ve-ptp/ sprouts, podocalyxin and moesin were disorganized, surrounding certain ECs cells and missing from others (Fig. 5a, right). Strikingly, ve-ptp/ sprouts did not enclose a lumen but often presented a cup-shaped structure (see stalk cell image in Fig. 5a, right). About 25% of ve-ptp/ ECs showed abnormally located podocalyxin (Fig. 5b) as determined in a double-blind assessment. At day 21 of differentiation, the WT sprouts displayed lumens encompassed by 2-3 ECs (Fig. 5c). ve-ptp/ sprouts on the other hand showed cup-shaped EC formations with disorganized podocalyxin/moesin immunostaining (Fig. 5d). Inspection of sprouts for i) continuous lumen formation along the sprout length, ii) the presence of occasional gaps or iii) cup-shaped sprouts, showed that only 30% of the ve-ptp/ sprouts contained a continuous lumen compared with 90% of WT sprouts (Fig. 5e). Importantly, the frequency of ECs with abnormal podocalyxin distribution decreased with decreased VEGF levels in the EB cultures (3 ng ml1; Fig. 5f). Furthermore, phosphorylation of Y658 in VE-cadherin was high in VEGF-treated ve-ptp/ EBs (20 ng ml1; Fig. 5g), and lowering the dose to 3 ng ml1 VEGF rescued pVE-cadherin levels to that of WT EBs (Fig. 5g). Figure 5h show quantification of podocalyxin distribution and pVE-cadherin levels in EBs treated with different concentrations of VEGF. In primary ECs, pY658 VE-cadherin levels increased with VEGF stimulation and decreased again when cells were treated with VEGFAng1 (Fig. 5j), implicating VE-PTP in regulation of pY658 VE-cadherin. Increased pY658 VE-cadherin was accompanied by reduced localization of VE-cadherin in ZO1-positive junctions in the ve-ptp/ sprouts (Fig. 5k). Combined, these data indicate that VE-PTP deficiency resulted in increased phosphorylation of VE-cadherin accompanied by polarization and lumen defects, in a manner that could be rescued by limiting VEGFR2 activation. To further define the role of Ang1/Tie2 in EC polarization and lumen formation, we treated WT EBs with a Tie2-extracellular domain soluble protein (sTie2/Fc) to neutralize endogenous Ang1 produced by the cultures. We hypothesized that blocking Ang1 using sTie2/Fc would suppress the capacity of VE-PTP to dephosphorylate VEGFR2. Indeed, podocalyxin was broadly distributed and surrounded ECs in sTie2/Fc-treated EBs, whereas untreated EB sprouts displayed polarized, apical distribution of podocalyxin (Fig. 6a). Furthermore, the sTie2/Fc-treated vessel sprouts showed reduced capacity to lumenize. In agreement with a role for VEGFR2 in this process, WT EBs treated with sTie2/Fc showed increased levels of both VEGFR2 and pVEGFR2, and the pVEGFR2/VEGFR2 ratio was elevated in the stalk region (Fig. 6c). These data show that neutralization of endogenously expressed Ang1 (Fig. 6e) by the sTie2/Fc phenocopied the effect of ve-ptp targeting, resulting in elevated pVEGFR2 activity and unpolarized vessels with lumen defects. Therefore, even though VEGF and Ang1 did not regulate the extent of formation of the trimeric complex (Fig. 2i), Ang1 appeared critical in junctional translocation of the complex. To provide in vivo confirmation of a role for VE-PTP in vessel lumen formation, we used WT and ve-ptp/ ESCs to establish teratomas in severe combined immunodeficient (SCID) mice. Teratomas become vascularized in part from the host and in part through differentiation of ECs from the ESCs34. As shown in Fig. 7a, vessels in ve-ptp/ teratomas displayed disorganized ECs with broadly distributed podocalyxin expression (Fig. 7a). Immunostaining for pVEGFR2 moreover demonstrated significant induction of VEGFR2 phosphorylation in CD31-positive ECs in ve-ptp/ teratomas (Fig. 7c). Co-immunostaining for pVEGFR2 and VE-PTP showed that ECs in perfused, lumenized vessels in WT teratomas expressed VE-PTP, but displayed very low levels of pVEGFR2. In contrast, disorganized and poorly perfused vessels in ve-ptp/ teratomas, which we confirmed lacked expression of VE-PTP, displayed prominent pVEGFR2 immunostaining (Fig. 7e). Omitting the primary antibody demonstrated the specificity of the immunostainings (Fig. 7e). There was furthermore a correlation between the extent of perfusion, the expression of VE-PTP and the ordered arrangement of VE-cadherin in the WT teratomas. In non-perfused vascular structures in the ve-ptp/ teratomas, the VE-cadherin pattern appeared fragmented (Fig. 7f). We also analysed VE-PTP expression in B16 F10 melanoma and identified a correlation between perfusion (functionality), VE-PTP expression and continuous VE-cadherin immunostaining on the one hand, and lack of perfusion, lack of VE-PTP expression and fragmented VE-cadherin on the other hand (Fig. 7g). Our data support a model in which Tie2-mediated suppression of VEGFR2 activity at EC junctions through VE-PTP is essential for formation of functional vessels. In pathological conditions characterized by excess VEGF production and faulty expression VE-PTP, ECs fail to polarize, which leads to a poorly functional vasculature with partially or completely collapsed lumen (Fig. 7h). We further explored VE-PTP's role in EC polarization and lumen formation in the zebrafish embryo. Formation of trunk intersomitic vessels (ISVs) in the zebrafish occurs through sprouting angiogenesis in a VEGF-dependent manner35,36,37,38. We first cloned the zebrafish VE-PTP orthologue, by searching for zebrafish sequences homologous to the mouse (m) VE-PTP catalytic domain. We identified a gene here denoted as ptp-rb, that we showed by in situ hybridization was expressed in ISVs during development (Supplementary Fig. S6). The corresponding protein is composed of fibronectin type III repeats repeats in the extracellular domain and a typical PTP catalytic intracellular domain. The catalytic domain of ptp-rb showed a striking degree of homology to murine VE-PTP with all catalytic PTP motifs presented. Two morpholinos (MOs) were used to specifically knock-down ptp-rb expression in the Tg(fli1a:EGFP)y1 transgenic background, where vascular endothelia express GFP39. The specificity of the ptp-rb MOs was verified by cloning of the resulting PCR fragment from MO-injected embryos (Supplementary Fig. S6d,e). In the 48-h post-fertilization embryos, control MO-treated ISVs displayed a characteristic chevron morphology and were lumenized. In contrast, in ptp-rb morphants, many ISVs had abnormal morphology and often lacked a continuous lumen (Fig. 8a-c). The number of embryos with ISV defects as well as the number of ISV defects per embryo significantly decreased by co-injecting the ptp-rb MO with human ptp-rb (ve-ptp) RNA, demonstrating that the vascular defects were due to loss of zebrafish ptp-rb function (Supplementary Fig. S7a-c). ptp-rb MO-treated ISVs failed to form a patent lumen (Fig. 8b); there was a twofold increase in lumen-less vessels/embryo when ptp-rb expression was suppressed (Fig. 8c). In accordance, double transgenic Tg(kdrl:EGFP)s843; Tg(gata1:dsRed)sd2 embryos showed flow in the control MO-treated embryos from the dorsal aorta (DA) through the ISVs to the dorsal longitudinal anastomosing vessel (DLAV), whereas embryos treated with ptp-rb MOs lacked dsRed-positive cells in the DLAV (Supplementary Fig. S7d); see also Supplementary Movies 1 and 2. This finding supports the notion that the majority of the ptp-rb-deficient ISVs lacked lumen, thereby preventing circulation through the ISV to the DLAV. In contrast, the ptp-rb-deficient DA contained blood cells, indicating that lumen formation in ISVs, which is established through sprouting angiogenesis, is distinct from lumenization of the DA during vasculogenesis. Cell counting in the Tg(fli1a:nEGFP)y7 transgenic background revealed no difference in the number of ISV ECs between ptp-rb MO-treated embryos and controls (data not shown). However, there was a significant increase in the number and density of long filopodia extending from the ISV ECs in the ptp-rb MO Tg(fli1a:nEGFP)y7 zebrafish (Fig. 8d). Immunostaining for podocalyxin in the control MO-treated fish embryos decorated the apical aspect of ECs, while ptp-rb MO-treated embryos showed a broad distribution and clear dysregulated polarity and lack of lumen (Fig. 8f). Immunostaining for zebrafish VE-cadherin (Cdh5) demonstrated a fragmented distribution and identified lumen-less vessels in the ptp-rb MO-treated embryos (Fig. 8g). Immunostaining for the tight junction protein ZO1 (Supplementary Fig. S7e) and the basement membrane component laminin, a marker for basal polarity (Suppelmentary Fig. S7f), further identified unpolarized, closed vessels in the absence of ptp-rb. Altogether, these data show that ptp-rb is essential for EC polarization and lumen formation in the zebrafish ISVs, demonstrating a conserved function of VE-PTP in zebrafish and mice.