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Published as doi: 10.1096/fj.07-8487com.
 (The FASEB Journal. 2007;21:3171-3183.)
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Activation of the orphan endothelial receptor Tie1 modifies Tie2-mediated intracellular signaling and cell survival

Hai Tao Yuan*,1, Shivalingappa Venkatesha*, Barden Chan{dagger}, Urban Deutsch{ddagger}, Tadanori Mammoto{dagger},2, Vikas P. Sukhatme{dagger}, Adrian S. Woolf§ and S. Ananth Karumanchi*,{dagger}

* Division of Molecular and Vascular Medicine, and

{dagger} Renal Division, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA;

{ddagger} Theodor Kocher Institut, Universität Bern, Bern, Switzerland; and

§ Nephro-Urology Unit, UCL Institute of Child Health, London, UK

1Correspondence: Division of Molecular and Vascular Medicine, and Renal Division, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Ave., RW663, Boston, MA 02215, USA. E-mail: hyuan{at}bidmc.harvard.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SPECULATIONS AND LIMITATIONS
 REFERENCES
 
A critical role for Tie1, an orphan endothelial receptor, in blood vessel morphogenesis has emerged from mutant mouse studies. Moreover, it was recently demonstrated that certain angiopoietin (Ang) family members can activate Tie1. We report here that Ang1 induces Tie1 phosphorylation in endothelial cells. Tie1 phosphorylation was, however, Tie2 dependent because 1) Ang1 failed to induce Tie1 phosphorylation when Tie2 was down-regulated in endothelial cells; 2) Tie1 phosphorylation was induced in the absence of Ang1 by either a constitutively active form of Tie2 or a Tie2 agonistic antibody; 3) in HEK 293 cells Ang1 phosphorylated a form of Tie1 without kinase activity when coexpressed with Tie2, and Ang1 failed to phosphorylate Tie1 when coexpressed with kinase-defective Tie2. Ang1-mediated AKT and 42/44MAPK phosphorylation is predominantly Tie2 mediated, and Tie1 down-regulates this pathway. Finally, based on a battery of in vitro and in vivo data, we show that a main role for Tie1 is to modulate blood vessel morphogenesis by virtue of its ability to down-regulate Tie2-driven signaling and endothelial survival. Our new observations help to explain why Tie1 null embryos have increased capillary densities in several organ systems. The experiments also constitute a paradigm for how endothelial integrity is fine-tuned by the interplay between closely related receptors by a single growth factor.—Yuan, H. T., Venkatesha, S., Chan, B., Deutsch, U., Mammoto, T., Sukhatme, V. P., Woolf, A. S., Karumanchi, S. A. Activation of the orphan endothelial receptor Tie1 modifies Tie2-mediated intracellular signaling and cell survival.


Key Words: angiopoietin • apoptosis • embryo • endothelial cells • Tie receptors


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SPECULATIONS AND LIMITATIONS
 REFERENCES
 
TIE1 AND TIE2 BELONG TO A SUBFAMILY OF VASCULAR tyrosine kinase receptors expressed predominantly in endothelial cells (1 , 2) . Targeted Tie1 disruption results in embryonic lethality due to edema, hemorrhage, and microvessel rupture (3 4 5) . Tie2 gene deletion causes embryonic death with endocardial defects, hemorrhaging, and impaired vascular network formation (4 5 6) . Embryos with targeted disruption of both Tie1 and Tie2 genes reveal an absolute requirement for Tie2 in the endocardium; as fetal development proceeds and the animal matures postnatally, the receptors become increasingly essential for capillary maintenance in diverse organs (7) . Tie1-deficient embryos contain hyperactive endothelial cells exhibiting a large number of extensions and filopodia projecting into the vessel lumen, and have increased capillary densities, suggesting that Tie1 may act as a brake during embryonic angiogenesis (5) ; the biological basis of this observation currently is unexplained.

Several angiopoietins (Ang1, Ang2, and Ang3/4) are Tie2 ligands, Ang1 and Ang2 being the most studied (1 , 2 , 8) . In endothelial cells, Ang1 induces Tie2 phosphorylation (9) ; Ang2 inhibits Ang1-induced Tie2 activation unless endothelial cells undergo prolonged exposure to higher concentrations of Ang2 (10) . Ang1 null mice have a similar phenotype as Tie2 null mutants (11) and Ang2 overexpression mimics most aspects of the Tie2 null phenotype with lethality in midgestation (9) . These data, therefore, are generally consistent with the idea that Ang1 is an agonist, and Ang2 an antagonist, of Tie2 activation. As recently reviewed, Ang1 has diverse actions on endothelial cells in vitro including enhanced migration, tube formation, and survival (12) . Biochemically, Ang1-mediated Tie2 phosphorylation causes phosphorylation of AKT threonine kinase and 42/44MAPK, both important in cell survival (13 14 15 16) .

Functional and biochemical studies of Tie1 have been hampered by the lack of a known physiological ligand. Inconsistent data were reported regarding Tie1 phosphorylation. Using Trk/Tie1 and Trk/Tie2 chimeric receptors, Tie2, but not Tie1, was activated by Trk ligand NGF (17) . Because Tie1 can be coimmunoprecipitated with Tie2, it was proposed that Tie1 and Tie2 heterodimerize (17) . However, in a separate study, chimeric c-fms/Tie1 receptor was phosphorylated by CSF-1, the ligand of c-fms (18) . Recently, COMP-Ang1 and native Ang1 were reported to induce Tie1 phosphorylation, with coexpression of Tie2 necessary for robust Tie1 activation (19) . Because a range of native and engineered Angs was reported to be unable to bind Tie1 (20 , 21) , it suggested that Tie1 activation was initiated indirectly by ligands binding to Tie2; the functional consequences of Tie1 phosphorylation and potential effects on downstream intracellular signaling were unexplored (19) .

We report here that Ang1 induces Tie1 phosphorylation in endothelial cells. Tie1 phosphorylation was, however, Tie2 dependent because 1) Ang1 failed to induce Tie1 phosphorylation when Tie2 was down-regulated in endothelial cells; 2) Tie1 phosphorylation was induced in the absence of Ang1 by either constitutively active Tie2 or a Tie2 agonistic antibody; and 3) Ang1 phosphorylated Tie1 when coexpressed with Tie2 and Ang1 failed to phosphorylate Tie1 when coexpressed with kinase-defective Tie2 in HEK 293 cells. We also demonstrate that Ang1-mediated AKT and 42/44MAPK phosphorylation is predominantly Tie2-mediated, and that Ang1-induced Tie1 activation down-regulates this pathway. Finally, based on a battery of in vitro and in vivo data, we show that a main role for Tie1 is to modulate blood vessel morphogenesis by virtue of its ability to down-regulate Tie2-driven endothelial survival.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 SPECULATIONS AND LIMITATIONS
 REFERENCES
 
Reagents
Chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless described otherwise. Recombinant human Ang1, anti-human Tie1, and Tie2 were purchased from R&D Systems (Minneapolis, MN, USA). Anti-phospho-AKT, total-AKT, total-42/44 MAPK, cleaved-caspase-3 (C-caspase-3), and phospho-Tie2(Tyr992) were purchased from Cell Signaling (Danvers, MA, USA). Antiphospho-42/44MAPK and anti-mouse CD31 were obtained from BD Biosciences (Bedford, MA, USA). Anti-GAPDH and ß-tubulin were obtained from Chemicon (Temecula, CA, USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA), respectively.

Cell cultures
HUVECs were purchased from Cascade Biologics, Inc. (Portland, OR, USA) and cultured in EBMTM-MV medium (Cambrex, East Rutherford, NJ, USA). HUVECs of <10 passages were used this study. Endothelioma cells isolated from Tie1+/+ and Tie1–/– or Tie2+/+and Tie2–/– embryos were cultured in DMEM with 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA), as described previously (22) .

Mouse Tie1, Tie2, mutant mouse Tie1-K/A, Tie2-K/A, human Tie2, and a constitutively active form of human Tie2 (T2A) -expressing constructs
The open reading frame of mouse Tie2 was amplified by PCR from IMAGE clone (ID 4195879) and subcloned into pcDNA 3.1 (Invitrogen), which contains a V5-Tag and a His-Tag at the C terminus of the protein. The open reading frame of mouse Tie1 (minus the predicted leader peptide) was amplified by PCR from cDNA prepared from mouse total RNA and subcloned into a pLNCX2-based retroviral expression vector that harbors, among other elements, a sequence encoding the Flag epitope and the EGFP gene. The open reading frame of human Tie2 was amplified by PCR from IMAGE clone 5228999 and subcloned into p-shuttle for generating adenoviral shuttle vector and adenovirus generated using the AdEasy system. Mouse Tie1-K/A and Tie2-K/A mutant constructs that have a lysine-to-alanine transition in the catalytic center of Tie1 and Tie2 tyrosine kinase domain were generated by PCR point mutagenesis. This point mutation leads to the inactivation of Tie1 and Tie-2 tyrosine kinase. Similarly, a constitutively active form of Tie2 (T2A) construct in which a C-to-T nucleotide transition that leads to an arginine-to-tryptophan substitution at position 849 (R849W) in the kinase domain of the receptor was generated by point mutagenesis, as previously reported (23) .

Establishment of Tie1, Tie2, and Tie1+Tie2-expressing HEK 293 cells
The pcDNA-Tie2 or the control plasmid (pcDNA3.1/V5-His/lacZ) was used to transfect HEK 293 cells using a calcium phosphate method. Cells that were stably transfected were selected using G418 at 800 µg/ml. Both the empty pLNCX-based vector and pLNCX-Tie1 plasmid were used to prepare retrovirus in HEK 293cells. The resultant virus was used to infect either the lacZ or Tie2 stable HEK 293 cells. Infected cells were sorted by FACS (using GFP and EGFP). In some experiments, Tie1-K/A and Tie2-K/A were transiently expressed in stable Tie2- or Tie1-expressing HEK 293 cells using lipofectamine-2000 (Invitrogen), and cells were cultured for 48 h before experiments.

Overexpression of a constitutively active form of Tie2 via Ad-T2A in HUVECs
HUVECs with ~80% confluence were infected with Ad-T2A at a low (1x108 PFU) and a high (5x108 PFU) titration, respectively. After 4 h, cells were washed twice with PBS; fresh medium was added and the infected cells were cultured for a total of 48 h before experiments.

Embryo generation and sample preparation
Timed matings were set up between male and female Tie1+/– mice (4) . The day of the vaginal plug was defined as embryonic day (E) 0.5. Animals were housed and bred according to requirements of the local government in Bern, Switzerland (permission number 04/05). Embryos were collected at E16.5, and a piece of the tail was taken for proteinase K-mediated lysis and subsequent genotyping by PCR. The embryos were either snap-frozen in liquid nitrogen for preparation of protein lysates or embedded in Tissue-Tek, OCT, then frozen on a 2-methylbutane (isopentane) dry ice bath for the preparation of cryosections. Genotyping PCRs were performed using the primers Tie-1 FW3 (CGA AGG GAT GGG AGA GAG AGC) and Tie1 I1 REV2 (TGA CGC TAT GAC GAC GAC GAT G) to detect the wild-type allele and using primers Tie-1 FW3 and PGK prom REV1 (TGT CAC GTC CTG CAC GAC GC) to detect the knockout allele. PCR protocol for both Tie-1 knockout and wild-type allele was as follows: 4 min, 94°C, followed by 35 cycles of 30 s, 94°C; 30 s, 60°C; and 60 s, 72°C, with a final elongation step of 4 min at 72°C.

Ang1 or agonistic Tie2 antibody stimulates Tie1 and Tie2 phosphorylation
Confluent HUVECs were stimulated with 200 ng/ml of Ang1 for up to 60 min or with 0–800 ng/ml Ang1 for 30 min to assess the time course and dose-response. For Tie receptor-expressing HEK 293 cells, 400 ng/ml Ang1 was used to stimulate the cells for 30 min. Polyclonal goat anti-human Tie2 (R&D Systems) was raised against the extracellular domain of human Tie2 protein. It was tested in the current study with both Western blot and immunoprecipitation (IP) Western blot to react specifically to Tie2. Confluent HUVECs was stimulated with 5 µg/ml anti-human Tie2 for 30 min. Normal goat IgG was used as control.

Matrigel capillary tube formation/regression assay
Capillary tube-like structures were generated overnight after HUVECs (2.5x104 cells/300 µl) were inoculated in 48-well plate precoated with 100 µl Matrigel (BD Biosciences) as described (24) . Structures so formed were left for 6 days without medium change for tubes undergoing regression. The total lengths of structures formed on day 1 and structures remaining on day 6 were derived using PCI Advanced Image software (Compix Inc. Imaging Systems, Sewickley, PA, USA) by measuring the total pixel numbers. The ratio of total pixel numbers of day 6 over day 1 was calculated and that of NC siRNA-transfected cells is valued as arbitral 1. Data are expressed as mean ± SD.

Apoptosis assay
HUVECs transfected with siRNAs were exposed to medium containing 0.1% FBS for 24 h to induce apoptosis. HUVECs, both those that remained on the Petri dishes and those in the supernatant, were collected and used for Western blot or IP-Western blot. In some experiments, cells that remained on the Petri dishes were used for TUNEL assay and C-caspase-3 immunofluorescent staining as described (25) .

Immunoprecipitation and Western blot
Cell or tissue lysates were prepared with RIPA buffer containing both proteinase/phosphatase inhibitors as described (26) . An equal amount of protein from each sample in 1 ml of RIPA buffer was used for IP. Primary antibodies used for IP include anti-human Tie1, Tie2, and anti-mouse Tie2 at 2–5 µg/sample. After overnight incubation at 4°C, the antigen-IgG complex was pulled down with protein G (Dynal Biotech, Great Neck, NY, USA) and subjected to electrophoreses. For Western blot, equal amounts of protein samples prepared from cultured cells, embryonic tissues, or IP samples were electrophoresed using NuPAGE Novex 3–8% Tris-acetate gel or NuPAGE Novex 4–12% Bis-Tris Gel (Invitrogen) and transferred onto PVDF membrane using semi-dry apparatus. The blots were blocked with TBST containing 0.1% Tween-20 and 5% nonfat milk for at least 1 h before primary antibodies were applied and incubated at 4°C overnight. The primary antibodies were detected using HRP-conjugated secondary antibodies and Super Signal West Pico/Femto Chemiluminescent Kits (Pierce Biotechnology, Rockford, IL, USA). The intensities of signal bands were analyzed using Image-J (NIH, Bethesda, MD, USA). Reverse IP for measuring Tie1 or 2 phosphorylation was performed using either normal mouse IgG2b or mouse monoclonal anti-P-Tyro (4G10, IgG2b) for precipitation and anti-Tie1 or -Tie2 for Western blot, respectively.

Immunofluorescent staining and confocal microscopy
HUVECs or cryosections were fixed with either 4% paraformaldyhyde (4°C) or methanol (–20°C) for 30 min. Anti-human Tie1 and anti-mouse CD31 were used as primary antibodies, followed by FITC, Texas red-conjugated anti-goat, or anti-rat IgG. Nuclei were counterstained with either propidium iodide (PI, Vector Laboratory, Burlingame, CA, USA) or To-Pro-3 (Invitrogen). Images were taken using a Bio-Rad MRC confocal microscope. TUNEL assay for cultured HUVECs was performed according to the manufacturer’s instruction (Roche Diagnostics, Nutley, NJ, USA). TUNEL-positive cells are counted under 20x magnification and expressed as (%) of total PI-positive cells. Six random fields were examined for each sample (n=6).

TUNEL assay and TUNEL/CD31 double staining
When TUNEL/CD31 double staining was performed to detect apoptotic endothelial cells in embryonic sections, the TUNEL assay was performed first and followed by CD31 staining, as described above (25) .

Statistics
Parameters were expressed as mean ± SD and groups compared using unpaired Student’s t test or by 1-way analysis of variance, followed by t tests.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 SPECULATIONS AND LIMITATIONS
 REFERENCES
 
Ang1 induces dose- and time-dependent Tie1 phosphorylation
After exposure of human umbilical vein endothelial cells (HUVECs) to 200 ng/ml of Ang1, increasing Tie1 phosphorylation was observed from 5 to 20 min; levels then fell but remained above baseline at 60 min (Fig. 1 A, B). Ang1-mediated Tie1 phosphorylation increased in a dose-dependent fashion between 50 and 800 ng/ml (Fig. 1C, D ). Ang1-induced Tie2 phosphorylation was assessed in parallel with Tie1, and we noted that Ang1-induced Tie1 phosphorylation was less prominent than that of Tie2 (Fig. 1A-D ). Reverse IP was performed to validate the specificity of antibodies used for Tie1 and Tie2 (Fig. 1E ).


Figure 1
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  		Figure 1. Ang1-induced Tie1 and Tie2 phosphorylation. Confluent HUVECs were stimulated with 200 ng/ml of Ang1 for up to 60 min (A, B) or with 0–800 ng/ml of Ang1 for 30 min (C, D) and immunoprecipated with Tie1 or Tie2 antibody. After IP, Western blot for phospho-tyrosine, total Tie1 and Tie2 proteins was performed. Representative blots of three independent experiments are depicted along with ratios of phospho/total proteins (mean±SD, n=3). Throughout this study, Tie1 was precipitated as a doublet, and it is the top band (fully membrane incorporated form of Tie1 as described; ref. 45 ) that is phosphorylated. E) Reverse IP was performed using either normal mouse IgG or mouse monoclonal anti-P-Tyro for precipitation and anti-Tie1 and Tie2 for Western blot with cell lysates of control and Ang1-stimulated HUVECs. Note that the specific Tie1 and Tie2 bands were detected only in anti-P-Tyro-precipitated samples but not in normal mouse IgG-precipitated lysates.

Ang1-induced Tie1 phosphorylation is Tie2 dependent
Similar to normal HUVECs, when negative control (NC) siRNA-transfected HUVECs were stimulated with 200 ng/ml Ang1, both Tie1 and Tie2 were phosphorylated (Fig. 2 A). In Tie1a siRNA-transfected HUVECs, Ang1-induced Tie1 phosphorylation disappeared while Tie2 phosphorylation appeared enhanced vs. NC siRNA-transfected cells (Fig. 2A ); the latter observation was explored in more detail with quantified data (see Fig. 6 below). In Tie2 siRNA-transfected HUVECs, Ang1-induced Tie2 phosphorylation was reduced by >95% and, strikingly, Ang1-induced Tie1 phosphorylation was abolished despite total Tie1 levels equivalent to NC-siRNA-transfected cells (Fig. 2A ). Hence, in HUVECs, Ang1-induced Tie1 phosphorylation is dependent on Tie2. Higher doses of Ang1 (up to 800 ng/ml) failed to induce Tie1 phosphorylation in Tie2 siRNA-transfected HUVECs, eliminating the trivial possibility that higher doses of Ang1 are required to stimulate Tie1 in HUVECs lacking Tie2 (Fig. 2B ). Tie1 was immunolocalized on plasma membranes in NC siRNA-transfected HUVECs and Tie1a siRNA transfection significantly reduced Tie1 expression. In Tie2 siRNA-transfected HUVECs, there was an impression of more prominent cytoplasmic Tie1 protein (Fig. 2C ), even though total Tie1 levels were similar to NC siRNA-transfected HUVECs (Fig. 2A, B ). In murine endothelioma cells isolated from wild-type and null mutant embryos, 200 ng/ml Ang1 induced Tie2 phosphorylation in Tie1+/+ and Tie1–/– cells, but Ang1 failed to induce Tie1 phosphorylation in Tie2–/– cells (Fig. 2D ); these results support our findings in HUVECs. Next, HEK 293 cell lines expressing murine Tie1, Tie2, or Tie1+Tie2 were established and used to further study the possible roles of Tie1 in modulating Ang1/Tie2 signaling in nonendothelial cells. Basal Tie1 phosphorylation was noted in Tie1 and Tie1+Tie2-expressing HEK 293 cells, with a higher level in the latter (Fig. 2E ). Contrary to the findings of a previous study (19) , no Ang1-induced Tie1 phosphorylation was detected in Tie1-expressing HEK 293 cells whereas Tie1+Tie2-expressing HEK 293 cells did show increased receptor phosphorylation (Fig. 2E ). Ang1-induced Tie2 phosphorylation was noted in both Tie2 and Tie1+Tie2-expressing HEK 293 cells (Fig. 2E ). These observations are consistent with our data from HUVECs (described above) that Ang1-induced Tie1 phosphorylation is Tie2 dependent.


Figure 2
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  		Figure 2. Ang1-induced, Tie2-dependent Tie1 phosphorylation. A) Ang1-induced Tie1 and Tie2 phosphorylation in NC, Tie1a, or Tie2 siRNA-transfected HUVECs. Tie1 protein was barely detectable in Tie1a siRNA-transfected cells whereas Tie2 protein was reduced by ~90% in Tie2 siRNA-transfected HUVECs. Ang1-induced Tie1 phosphorylation disappeared in Tie2 and Tie1a siRNA-transfected HUVECs. Ang1-induced Tie2 phosphorylation was reduced by >95% in Tie2 siRNA-transfected HUVECs and appeared to be enhanced in Tie1a siRNA-transfected HUVECs. B) Doses of Ang1 of up to 800 ng/ml failed to induce Tie1 phosphorylation in Tie2 siRNA-transfected HUVECs. C) HUVECs transfected with NC, Tie1a, or Tie2 siRNA underwent immunocytochemistry for Tie1 (green), nuclei counterstained with PI (red). Tie1 was detected in plasma membrane and cytoplasmic patterns in NC siRNA-transfected HUVECs but was barely detectable in Tie1a siRNA-transfected cells; in Tie2 siRNA-transfected HUVECs, the plasma membrane signal for Tie1 appeared to be attenuated. D) Ang1-induced Tie1 and Tie2 phosphorylation in murine endothelioma cells isolated from Tie1+/+ (wild-type, WT) and Tie1–/– (null mutant, NU) or Tie2+/+ and Tie2–/– embryos. Tie1 and Tie2 proteins were not detected in Tie1–/– or Tie2–/– endothelioma cells. 200 ng/ml Ang1 phosphorylated Tie1 and Tie2 in both Tie1+/+ and Tie2+/+ endothelioma cells; Ang1 could barely induce Tie1 phosphorylation in Tie2–/– endothelioma cells, whereas Tie2 phosphorylation was not affected in Tie1–/– endothelioma cells. E) Tie1, Tie2, or Tie1+Tie2-expressing HEK 293 cells were stimulated with 400 ng/ml of Ang1 for 30 min and lysates were subjected to IP-Western blot. Ang1-induced Tie2 phosphorylation in both Tie2- and Tie1+Tie2-expressing HEK 293 cells. Basal Tie1 phosphorylation was noted in both Tie1- and Tie1+Tie2-expressing HEK 293 cells, with a higher phospho-Tie1 level in Tie1+Tie2-expressing HEK 293 cells. Ang1-induced Tie1 phosphorylation in Tie1+Tie2 but not in Tie1-expressing HEK 293 cells.


Figure 3
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  		Figure 3. Tie2-mediated Tie1 phosphorylation. A) Elevated Tie1 phosphorylation in HUVECs overexpressing a constitutively active form of Tie2. 48 h after infection, protein lysates from HUVECs infected without (C) or with adenovirus-expressing GFP (G), low (L) or high (H) titers of constitutively active form of human Tie2 (T2A) were subjected to IP-Western blot for phospho-tyrosine, total Tie1 and Tie2 proteins. Dose-dependent increases of phospho-Tie2 were recorded in Ad-T2A-infected HUVECs, accompanied by dose-dependent increases of phospho-Tie1. B) Tie1 phosphorylation in HUVECs stimulated with agonistic Tie2-specific antibody. Confluent HUVECs were stimulated with normal goat IgG (Con) or goat-anti-human Tie2 (a-Tie2) antibody (5 µg/ml) for 30 min, and protein lysates were subjected to IP-Western blot for phospho-tyrosine, total Tie1 and Tie2 proteins. Tie2 agonistic antibody stimulated phospho-Tie2 level compared with HUVECs treated with normal goat IgG. When Tie1 proteins were precipitated, Tie1 protein was phosphorylated in HUVECs treated with Tie2-specific agonistic antibody. Note that the phospho-Tie1 band aligned well with the top band of the total Tie1 doublets (top arrowhead). C) Tie1 phosphorylation is mediated via Tie2 tyrosine kinase. Stable Tie1-expressing HEK 293 cells were transiently coexpressed with WT or a kinase inactive form of Tie2 (Tie2-K/A). Stable Tie2-expressing HEK 293 cells were transiently transfected with WT or a kinase inactive form of Tie1; the cells were stimulated with 400 ng/ml Ang1 for 30 min, then protein lysates were subjected to IP with anti-Tie1 or Tie2 and blotted for phospho-tyrosine. Ang1 induced Tie1 and Tie2 phosphorylation in stable Tie2-expressing cells coexpressing Tie1 or Tie1-K/A. Ang1 induced Tie1 and Tie2 phosphorylation in stable Tie1-expressing cells when WT Tie2 was coexpressed, but not Tie2-K/A, as no phospho-Tie2 was detectable in those cells.


Figure 4
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  		Figure 4. Ang1-induced AKT and 42/44MAPK phosphorylation. A–D) Ang1 induced AKT and 42/44MAPK phosphorylation in siRNA-transfected HUVECs. HUVECs transfected with NC, Tie1a, Tie1b, and Tie2 siRNA were stimulated with Ang1 (200 ng/ml) for 30 min. Phospho/total AKT (A, B) and phospho/total 42/44MAPK (C, D) levels were analyzed with Western blot; depicted blots (A, C) are representative of three independent experiments and data are expressed as ratios (mean±SD, n=3) of phospho/total AKT (B) and phospho/total 42/44MAPK (D). The control level of NC siRNA-transfected cells is defined as an arbitral value of 1. Ang1 induced AKT (A) and 42/44MAPK (C) phosphorylation in NC siRNA-transfected HUVECs and Ang1-induced AKT (A) and 42/44MAPK (C) phosphorylation were abolished in Tie2 siRNA-transfected HUVECs vs. NC siRNA-transfected cells. A significantly elevated ratio of phospho/total AKT (P<0.05, B) and phospho/total 42/44MAPK (P<0.01, D) was observed in Tie1a and Tie1b siRNA-transfected HUVECs. E) Ang1-induced AKT and 42/44MAPK phosphorylation in Tie2, and Tie1+Tie2-expressing HEK 293 cells. Quantitative data shown in panels F (phospho/total AKT ratio) and G (phospho/total 42/44MAPK ratio) demonstrated a modest but significant reduction of Ang1-induced phospho/total AKT (P<0.05, n=3) and phospho/total 42/44MAKP ratios (P<0.05, n=3) in Tie1+Tie2-expressing HEK 293 cells vs. cells expressing Tie2 alone.


Figure 5
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  		Figure 5. Capillary tube regression in Matrigel and endothelial apoptosis under low serum conditions. A). The ratios of total lengths of remaining capillary tubes on day 6 over tubes formed on day 1 were calculated; note that Tie1a and Tie1b siRNA-transfected HUVECs had significantly more tubes on day 6 (P<0.01, n=6), whereas ~60% fewer tubes were observed in Tie2 siRNA-transfected HUVECs. NC, Tie1a, and Tie1b siRNA-transfected HUVECs were cultured with 0.1% FBS for 24 h, and adherent and floating cells were subjected to Western blot for C-caspase-3 (B, C). The blot depicted is representative of three independent experiments. C-caspase-3 levels were significantly lower in both Tie1a and Tie1b siRNA-transfected HUVECs vs. NC siRNA-transfected cells (**P<0.01, n=3). D) Percentages vs. total cells of TUNEL or C-caspase-3-positive cells; note the significantly decreases in TUNEL and C-caspase-3-positive cells in HUVECs transfected with Tie1a and Tie1b siRNA vs. NC HUVECs (mean±SD, n=6; *P<0.05 or **P<0.01, n=6).


Figure 6
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  		Figure 6. Enhanced Tie2 phosphorylation and downstream signaling in Tie1 siRNA-transfected endothelial cells. A) Ang1-induced Tie2 phosphorylation in Tie1 siRNA-transfected HUVECs. HUVECs transfected with NC, Tie1a, or Tie1b siRNA were stimulated with 200 ng/ml Ang1 for 30 min and protein lysates were subjected to Western blot with a specific phospho-Tie2 (Tyr992) antibody. Ang1-induced Tie2 phosphorylation was enhanced in both Tie1a and Tie1b siRNA-transfected cells; the blot is representative of three independent experiments. B) Ang1-induced Tie2 phosphorylation in NC, Tie1a, and Tie1b siRNA-transfected HUVECs shown as phospho/total-Tie2 ratio (mean±SD, n=3, the ratio of the cells without Ang1 stimulation is defined as an arbitrary value of 1). Ang1 increases the phospho/total Tie2 ratio in NC, Tie1a, and Tie1b siRNA-treated cells (**P<0.01); in both Tie1a and Tie1b siRNA-transfected HUVECs, the level of phospho/total Tie2 ratios was ~2-fold higher than that in NC siRNA-transfected HUVECs (#P<0.05). C) HUVECs transfected with NC, Tie1a, Tie1b, or Tie2 were exposed to low serum conditions (0.1% FBS) for 24 h, and protein lysates were subjected to IP with Tie2 and blotted with total and phospho-Tie2 (Tyr992). The same cell lysates were probed with anti-GAPDH for loading control. Tie2 phosphorylation was enhanced in both Tie1a and Tie1b siRNA-transfected cells after exposure to low serum conditions for 24 h; the depicted blot is representative of three independent experiments. D) Ratios of phospho/total-Tie2 of NC, Tie1a, Tie1b, and Tie2 under low serum conditions (mean±SD, n=3) are shown. Note ~1.5- and 1.6-fold increase of phospho/total Tie2 in Tie1a and Tie1b siRNA-transfected (*P<0.05) vs. NC siRNA-treated cells. A decreased Tie2 phosphorylation by >75% was also found in Tie2 siRNA-transfected HUVECs. E–H) Western blots (representative of 3 independent experiments) for phospho- and total-AKT (E) and -42/44MAPK (G), and phospho/total ratios (mean±SD, n=3; F, H) in HUVECs transfected with NC, Tie1a, Tie1b, or Tie2 siRNA under low serum conditions for 24 h. Note a significantly elevated ratio in the Tie1a and Tie1b siRNA-transfected HUVECs vs. NC and Tie2 siRNA-transfected cells (P<0.05).

Tie2 tyrosine kinase mediates Tie1 phosphorylation
When constitutively active Tie2 (T2A) was overexpressed in HUVECs using Ad-T2A, a dose-dependent increase of both total- and phospho-Tie2 was observed (Fig. 3 A). Meanwhile, a dose-dependent increase of phospho-Tie1 was noted even though the total Tie1 level remained the same in those cells (Fig. 3A ). The anti-Tie2 antibody used in the current study was raised against the extracellular domain of human Tie2. Using both Western blot and IP-Western blot, we confirmed that this antibody has no cross-reactivity to Tie1. Unexpectedly, when confluent HUVECs were exposed to this antibody (5 µg/ml) for 30 min, an increased Tie2 phosphorylation was noted; meanwhile, the Tie2 phosphorylation was accompanied by Tie1 phosphorylation (Fig. 3B ), indicating that, in the absence of Ang1, Tie2 phosphorylation could lead to Tie1 phosphorylation. Finally, to demonstrate that it is the Tie2 tyrosine kinase activity that is primarily responsible for Tie1 tyrosine phosphorylation, stable Tie1-expressing HEK 293 cells were transiently transfected with Tie2 or Tie2-K/A; in these cells, a basal level of Tie1 phosphorylation was observed; Ang1-induced Tie1 (additional) phosphorylation was observed in stable Tie1-expressing HEK 293 cells coexpressing Tie2, but not Tie2-K/A (Fig. 3C ). On the other hand, Ang1-induced Tie2 phosphorylation was noted in stable Tie2-expressing HEK 293 cells coexpressing either Tie1 or Tie1-K/A; in the same sets of cells, both Tie1 and Tie1-K/A were phosphorylated by Ang1 (Fig. 3C ).

Tie1 modulates Ang1/Tie2 intracellular signaling
Ang1-induced phosphorylation of AKT and 42/44MAPK signaling was compared in HUVECs in which Tie1 or Tie2 was down-regulated (Fig. 4 A–D). Ang1 induced AKT phosphorylation in NC siRNA-transfected HUVECs; this elevated AKT phosphorylation was abolished in Tie2 siRNA-transfected HUVECs. Ang1-induced AKT phosphorylation was significantly more prominent in HUVECs transfected with both Tie1a and Tie1b siRNA (Fig. 4A, B ). Similarly, we found that Ang1 increased 42/44 MAPK phosphorylation in NC siRNA-transfected HUVECs, and this response was also abolished in Tie2 siRNA-transfected HUVECs. In Tie1a and Tie1b siRNA-transfected HUVECs, significantly more prominent increases in phospho-42/44MAPK were noted after Ang1 exposure (Fig. 4C, D ). Collectively, these results support the conclusions that Ang1-induced AKT and 42/44MAPK phosphorylation in cultured endothelial cells are primarily Tie2 mediated and that Ang1-induced Tie1 activation appears to down-regulate Ang1/Tie2 signaling. Ang1-induced AKT and 42/44MAPK phosphorylation were also compared between Tie2 and Tie1+Tie2-expressing HEK 293 cells (Fig. 4F ), and modest but significant reductions of Ang1-induced AKT (Fig. 4G ) and 42/44MAPK (Fig. 4H ) phosphorylation were noted in Tie1+Tie2-expressing HEK 293 cells vs. Tie2-expressing HEK 293 cells, supporting the notion, derived from the HUVEC experiments, that Ang1-associated Tie1 activation down-regulates Ang1/Tie2 signaling.

Tie1 diminishes survival of endothelial cells in tissue culture
Next we used an in vitro model of capillary tube regression to begin to address the biological significance of the foregoing biochemical observations. The total length of tubes formed on day 1 from HUVECs transfected with NC, Tie1a, Tie1b, or Tie2 siRNAs and the total length of remaining tubes on day 6 in each group were analyzed and expressed as the ratio of the length of remaining tubes on day 6 over that on day 1 (Fig. 5 A). Tie1a and Tie1b siRNA-transfected HUVECs had ~3.7- and 2.8-fold more remaining tubes vs. NC siRNA-transfected cells (P<0.01). On the other hand, Tie2 siRNA-transfected HUVECs had ~60% fewer remaining tubes than NC-siRNA-transfected cells (P<0.05). Using Western blot, significantly lower C-caspase-3 levels were found in Tie1a and Tie1b siRNA-transfected HUVECs than in NC siRNA-transfected cells (Fig. 5B, C ). Using an assay of low serum-induced programmed cell death (Fig. 5D ), we recorded a significantly lower proportion of TUNEL-positive apoptotic cells in both Tie1a (20.0±1.5%) and Tie1b (26.3±1.3) siRNA-transfected HUVECs vs. endothelial cells transfected with NC siRNA (36.3±4.1). Similarly, a lower percentage of C-caspase-3-positive cells was noted in Tie1a (15.4±4.8) and Tie1b (20.9±2.1) siRNA-transfected HUVECs vs. control transfectants (50.0±4.0) (Fig. 5D ).

Tie1 down-regulates Ang1/Tie2 signaling
From data depicted in Fig. 2A , there appeared to be enhanced Ang1-induced Tie2 phosphorylation in Tie1a siRNA-transfected HUVECs. To clarify whether Tie1 phosphorylation regulates Tie2 phosphorylation, experiments were repeated using both Tie1a and Tie1b siRNA-transfected HUVECs; anti-phospho-Tie2 (Tyr992) antibody was used to specifically demonstrate Tie2 endodomain tyrosine (Tyr992) phosphorylation. Significantly enhanced (2-fold, P<0.05) Ang1-induced Tie2 phosphorylation was observed in both Tie1a and Tie1b siRNA-transfected HUVECs vs. NC-siRNA-transfected HUVECs (Fig. 6 A, B). After exposure to low serum medium for 24 h, HUVECs transfected with Tie1a and Tie1b siRNA maintained a significantly higher level (~2.3- and 2.1-fold) of phospho-Tie2 (P<0.05 for both Tie1a and Tie1b vs. NC siRNA-treated cells, Fig. 6C, D ). In addition, HUVECs transfected with Tie1a and Tie1b siRNA had significantly higher levels of phospho/total AKT (Fig. 6E, F ) and phospho/total 42/44MAPK (Fig. 6G, H ) vs. NC siRNA- or Tie2 siRNA-transfected cells.

Increased endothelial densities in Tie1-deficient embryos correlate with decreased C-caspase-3 and increased levels of phospho-AKT
Having made the aforementioned observations linking Tie1 to Tie2 intracellular signaling and cell survival in endothelial cells in vitro, we proceeded to assess whether these observations had any relevance to endothelial biology in vivo. Similar to previous reports (4) , we observed that embryonic day 16.5 Tie1–/– embryos had marked peripheral edema (data not shown). Using immunohistochemistry for CD31 (an endothelial marker), more extensive networks of vessels were noted in Tie1–/– vs. wild-type embryos, especially in the liver (Fig. 7 A, B), diaphragm, bone, and dermis of the skin (data not shown). Apoptotic endothelial cells in vivo were demonstrated by TUNEL/CD31 immunostaining; there appeared to be more dying endothelial cells in liver (Fig. 7C, D ) and skin (data not shown) of Tie1+/+ embryos vs. Tie1–/– embryos. Pairs of embryos harvested on day 16.5 of gestation from individual litters (Fig. 7E : litter 1, Tie1–/–and Tie1+/+; litter 2, Tie1–/– and Tie1+/–; and litter 3, Tie1+/– and Tie1+/+) were used for Western blot for CD31 and Tie1, confirming that Tie1 was not detectable in Tie1–/– embryos and that the Tie1 level was clearly lower in Tie1+/– embryos vs. Tie1+/+ embryos (Fig. 7E ). Levels of CD31 were in the following order: Tie1–/– > Tie1+/– > Tie1+/+ embryos. IP-Western blot of Tie1+/+ and Tie1–/– embryos harvested on day 16.5 of gestation confirmed that phospho-Tie2 was increased in Tie1 null mutants, while total Tie2 levels were similar to wild-type (Fig. 7F ). Pairs of embryos from individual litters (Fig. 7G ) were also used for Western blot for total/phospho-AKT, 42/44 MAPK, and C-caspase-3; although no differences in phospho- and total 42/44MAPK were found, there was a prominent increase of phospho-/total AKT in null mutants vs. Tie1 heterozygous or wild-type embryos (Fig. 7G ). In addition, C-caspase-3 levels appeared to be lower in Tie1–/– embryos than in the other two genotypes (Fig. 7G ).


Figure 7
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  		Figure 7. CD31 and TUNEL staining as well as levels of CD31, Tie1/2, AKT, 42/44MAPK, and C-caspase-3 proteins in Tie1+/+, Tie1+/–, and Tie1–/– embryos. Immunofluorescent staining of CD31 (red) counterstained with To-Pro-3 (blue) of nuclei and of E16.5 embryonic liver tissues from Tie1+/+ (A) and Tie1–/– (B) embryos. Note the more prominent vasculature in the null mutants. Coimmunofluorescent staining of CD31 (red) and TUNEL (green) of E16.5 embryonic liver tissues from Tie1+/+ (C) and Tie1–/– (D) embryos; dying endothelial cells appeared more plentiful in wild-type vs. Tie1–/– embryos. E) Levels of Tie1 and CD31 in Tie1+/+, Tie1+/–, and Tie1–/– embryos. Western blots of three pairs of gestation day 16.5 embryos from three individual litters (litter 1: Tie1–/– and Tie1+/+; litter 2: Tie1–/– and Tie1+/–, litter 3: Tie+/– and Tie1+/+). Tie1 protein was not detectable in the Tie1–/– embryo and markedly reduced in the Tie1+/– embryo. Levels of CD31 were in the following order: Tie1–/– >Tie1+/– > Tie1+/+ embryos. F) IP-Western blot was performed with one pair of Tie1–/– and Tie1+/+ embryos. Level of total Tie2 was similar but increased phospho-Tie2 was noted in Tie1–/– embryo vs. Tie1+/+ embryo. G) Levels of phospho/total AKT and phospho/total 42/44MAPK in Tie1+/+, Tie1+/–, and Tie1–/– embryos. Note that phospho-AKT levels were higher and C-caspase 3 levels lower in Tie1–/– embryos than in Tie1+/– and Tie1+/+ embryos.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
SPECULATIONS AND LIMITATIONS
 REFERENCES
 
As outlined earlier, a role for Tie1 in blood vessel morphogenesis has emerged from mutant mouse studies, and it was recently demonstrated that certain angiopoietin (Ang) family members activate Tie1 (19) . We report here that Ang1 elicits Tie1 phosphorylation; Tie1 phosphorylation is, however, Tie2 dependent. We hypothesized that Tie1 activation would be associated with changes in Tie2-mediated intracellular signaling pathways and that such changes would correlate with an alteration in critical cell behavior, namely, survival. We demonstrated that Ang1-mediated AKT and 42/44MAPK phosphorylation is predominantly Tie2-mediated and that Tie1 down-regulates this pathway. Finally, based on a battery of in vitro and in vivo data, we show that a main role for Tie1 is to modulate blood vessel morphogenesis by virtue of its ability to down-regulate Tie2-driven downstream signaling and endothelial survival. Our new observations explain why Tie1 null embryos have increased capillary densities in several organ systems.

Ang1 induces Tie1 phosphorylation in endothelial cells
Recently, COMP-Ang1 and the native Ang1 were both reported to induce Tie1 phosphorylation (19) . We confirmed here that human Ang1 indeed induces Tie1 as well as Tie2 phosphorylation in HUVECs in a dose- and time-dependent manner. Differential intensities of Ang1-induced Tie1 and Tie2 phosphorylation were noted in our study even though both receptors were activated by Ang1, with a similar time-response course. Ang1-induced Tie1 phosphorylation was also observed in other endothelial cells, such as EAhy926 cells and human dermal microvascular endothelial cells (data not shown).

Ang1-induced Tie1 phosphorylation is Tie2 dependent
Ang1 was discovered as a ligand for Tie2, not Tie1, due to the lack of binding capacity of Ang1 to Tie1 (20) , which indicates that Ang1-induced Tie1 phosphorylation may be Tie2 dependent. Our data from both Tie2-down-regulated HUVECs and Tie2–/– murine endothelioma cells demonstrate that Tie1 activation is indeed Tie2 dependent (Fig. 2) . In other studies, COMP-Ang1 induced Tie1 phosphorylation in Tie1-expressing HEK 293 cells (19) and CSF-1 induced c-fms/Tie1 phosphorylation in NIH3T3 cells, suggesting a Tie2-independent Tie1 activation in those nonendothelial cells (18) . Endothelial-specific phenomena might be an oversimplified explanation for our inconsistent findings, as we failed to detect any Ang1-induced Tie1 phosphorylation in Tie1-expressing HEK 293 cells, which have a low level of basal phospho-Tie1. Perhaps inconsistencies could be explained by the extremely high doses of the more potent COMP-Ang1 (which has also been noted to form fewer multimers) used in a previous study (19) . As similar intensities of Tie1 phosphorylation were noted by 200 ng/ml of COMP-Ang1 and 600 ng/ml native Ang1 (Fig. 5 of the original report), 600 ng/ml COMP-Ang1 used for inducing Tie1 activation in that study is equivalent to 1800 ng/ml native Ang1. This is 4.5-fold higher than the dose used (400 ng/ml) in our study (19) . In our study, Ang1-induced Tie1 phosphorylation was only noted in HEK 293 cells coexpressing Tie1 and Tie2, which is consistent with our data from HUVECs.

Tie2 tyrosine kinase phosphorylates Tie1
The mechanism by which Tie1 is phosphorylated after Ang1 stimulation is still unknown. As Tie1 has been reported to heterodimerize with Tie2 at both basal and activated conditions (17 , 19) , we propose that Ang1-induced Tie1 phosphorylation is mediated via Tie2 tyrosine kinase activity. Several approaches have been used to prove our hypothesis. Overexpressing a constitutively active form of Tie2 using Ad-T2A in HUVECs or stimulating Tie2 of HUVECs with a Tie2-specific agonistic antibody results in increased Tie2 phosphorylation. When cells were transfected with a construct that expresses a constitutively active form of Tie2, phospho-Tie1 was noted in those cells and the phospho-Tie1 level appeared to correlate with the level of phospho-Tie2 (Fig. 3A ). These findings support the notion that Tie2 phosphorylation mediates Tie1 phosphorylation. Furthermore, Ang1 fails to phosphorylate Tie1 when Tie2-K/A, with an inactive form of Tie2 tyrosine kinase, is coexpressed with Tie1; on the other hand, Ang1 phosphorylates Tie1-K/A, an inactive form of Tie1-K/A, when Tie2 is coexpressed. These new findings provide the most convincing evidence so far that it is the Tie2 tyrosine kinase activity that phosphorylates Tie1.

Tie1 modulates Ang1/Tie2-mediated signaling and functions in cultured endothelial cells
Ang1 has been reported to play multifunctional roles in endothelial cells, presumably mediated via Tie2. Numerous studies have shown that Ang1 promotes endothelial migration (28 , 29) , reduces capillary permeability, and inhibits endothelial inflammatory responses (30 , 31) . Ang1 is a potent survival factor, with Ang1-induced AKT and 42/44MAPK phosphorylation implicated in protecting endothelial cells from apoptosis (14 15 16 , 32) . We found that Ang1-induced phosphorylation of AKT and 42/44MAPK is abolished in Tie2-down-regulated HUVECs, suggesting a predominant role of Tie2 in activating these signaling pathways. We made the unexpected observation that Ang1-induced AKT and 42/44MAPK phosphorylation are enhanced in Tie1-down-regulated HUVECs, suggesting that Tie1 activation somehow down-regulates such signaling. These findings appear inconsistent with a previous report that, via ligand-mediated dimerization, activated c-fms/Tie1 resulted in AKT activation and subsequent protection of cells from apoptosis; however, nonendothelial (NIH3T3) cells were used in that study (18) . The mechanism of the paradox that Tie1 activation down-regulates AKT and 42/44MAPK is unknown. If Tie1/Tie2 dimers are formed upon Ang1 stimulation, perhaps they are less efficient than Tie2 homodimers in mediating Ang1 signaling in HUVECs, including AKT and 42/44MAPK phosphorylation. When Tie1 is down-regulated in HUVECs, the majority of Ang1 signaling will be mediated via the presumably more efficient Tie2 homodimers, which in the end leads to enhanced overall Ang1 signaling. Our hypothesis is further supported by findings that Ang1-induced Tie2 phosphorylation is enhanced in HUVECs when Tie1 is down-regulated (Fig. 6A, B ). Nevertheless, the possibility that Tie1 activation inhibits Tie2 signaling via an unidentified Tie1 downstream molecule cannot be ruled out, and further studies are needed.

Functionally, Tie1-down-regulated HUVECs demonstrated relative resistance for apoptosis based on our in vitro data. This resistance to apoptosis appears to occur by maintaining a higher level of phospho-Tie2 in those cells, which subsequently induces a higher level of phospho-AKT and -42/44MAPK in Tie1 siRNA-transfected HUVECs. The fact that ablating Tie2 by siRNA showed an increased capillary tube regression supports the notion that the enhanced tube survival due to Tie1 knockdown may depend on Tie2. Our data are consistent with a previous report that blocking Tie2 signaling with either RNAi or overexpression of a kinase-dead Tie-2 results in the loss of endothelial cell viability due to a blockage to the AKT pathway (33) . The mechanism of the higher phospho-Tie2 level in Tie1 down-regulated HUVECs under low serum conditions is still unknown. Ang1 is primarily expressed by periendothelial cells, not endothelial cells (11 , 20 , 34) ; on the other hand, Ang2, a context-dependent agonist/antagonist of Tie2, is selectively expressed by endothelial cells, including HUVECs (9 , 35 , 36) . Ang2 has recently been reported as a protective factor in stressed endothelial cells by activating Tie2/AKT signaling (34) . Therefore, the potential role of Ang2 in enhanced Tie2 phosphorylation should be explored further.

Tie1 modulates embryonic blood vessel morphogenesis by diminished endothelial survival
Do our in vitro biochemical and functional observations have any relevance to blood vessel biology in vivo? In a morphological analysis of Tie1 null mice, Patan reported that midgestation embryos had more endothelial cells with an activated phenotype; there was also strikingly increased vascular density in diverse organs (brain, kidney, skin, lung, intestines) (5) . We confirmed the impression of increased microvessel densities in various organs of embryonic day 16.5 Tie1 null mutant mice. Patan favored the explanation that the lack of Tie1 had a primary effect on capillary remodeling rather than numbers because endothelial cell mass was not increased, at least as assessed by FLK1 and PECAM (CD31) Northern blots. Our own CD31 Western blot data suggest that endothelial mass is increased in Tie1–/– mice. Furthermore, at the same gestational stage, null mice have increased levels of phospho-AKT and decreased C-caspase-3. Our in vitro data (based on murine endothelioma cells isolated from wild-type and null mutant Tie1 embryos, as well as HUVECs), together with the in vivo observations in Tie1 mutant embryos, are consistent with the notion that, prenatally, a major role for Tie1 is to prevent excessive endothelial cell survival generated by Ang1/Tie2 signaling. In this study, we also observed that, in vitro, Tie1 deficiency is associated with increased endothelial migration (data no shown), which may provide another explanation, in addition to enhanced survival, as to why Tie1 null embryos appear to have a more extensive microvasculature containing activated endothelial cells (5) . On the other hand, Tie1 may play other roles in more mature vasculature because endothelial cells lacking Tie1 are excluded from vessels of postnatal mice (7) .


   SPECULATIONS AND LIMITATIONS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SPECULATIONS AND LIMITATIONS
REFERENCES
 
Our findings may have profound significance for understanding the pathogenesis of vascular disorders. Since the overall dose-response curve of Tie1 phosphorylation in response to physiological Ang1 is less robust than that of Tie2, the most likely diseases where Tie1 may regulate Tie2 signaling are those where Tie1 is overexpressed vs. Tie2. One such clinical scenario is atherosclerosis; in fact, Tie1 is markedly overexpressed in atherosclerosis-prone niches (37) . Therefore, high levels of Tie1 in the areas of disturbed flow may attenuate physiological Tie2 signaling and contribute to the pathogenesis. Another clinical scenario illustrating a functional role for Tie1 may be in tumor angiogenesis because Tie1 is up-regulated by hypoxia and VEGF, two mediators frequently present in the tumor milieu (38) . Clinical studies do indeed indicate Tie1 up-regulation in renal carcinomas (39) . Therefore, Tie1 may serve as a valuable target when devising therapies to perturb tumor vasculature. More generally, the experiments described here constitute a paradigm as to how endothelial integrity is fine-tuned by an interplay between closely related receptors that can be activated by a single growth factor ligand. Such a concept is reminiscent of the modulation of VEGF signaling via FLK1/VEGFR2, which can be elicited by FLT1/VEGFR1; in this case, FLT1 can modify signaling by intermolecular cross-talk with FLK1 (40 41 42 43 44) . Although in normal development, the VEGF axis is critical for initiation and proliferation of capillary endothelial cells, and the Ang1 axis plays later roles, it is notable that each phase of vascular differentiation/maturation will be controlled by critical interactions of pairs of related ligand receptors in response to a single growth factor, VEGF or Ang1.


   ACKNOWLEDGMENTS
 
This work was funded by National Institutes of Health grants to S.A.K. and V.P.S., and by a Kidney Research UK (formerly NKRF) project grant to H.T.Y. and A.S.W. We thank members of the Karumanchi and Sukhatme laboratories for helpful discussions.


   FOOTNOTES
 
2 Current address: Union Memorial Hospital, Department of Surgery, 200 E. 33rd. St. Bldg., Ste. #429, Baltimore, MD 21218, USA.

Received for publication February 28, 2007. Accepted for publication April 12, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SPECULATIONS AND LIMITATIONS
 REFERENCES
 

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