VEGF Induces Tie2 Shedding via a Phosphoinositide 3-Kinase/Akt–Dependent Pathway to Modulate Tie2 Signaling

【摘要】  Objective— Tie2 and its ligands, the angiopoietins (Ang), are required for embryonic and postnatal angiogenesis. Previous studies have demonstrated that Tie2 is proteolytically cleaved, resulting in the production of a 75-kDa soluble receptor fragment (sTie2). We investigated mechanisms responsible for Tie2 shedding and its effects on Tie2 signaling and endothelial cellular responses.

Methods and Results— sTie2 bound both Ang1 and Ang2 and inhibited angiopoietin-mediated Tie2 phosphorylation and antiapoptosis. In human umbilical vein endothelial cells, Tie2 shedding was both constitutive and induced by treatment with PMA or vascular endothelial growth factor (VEGF). Constitutive and VEGF-inducible Tie2 shedding were mediated by PI3K/Akt and p38 MAPK. Tie2 shedding was blocked by pharmacological inhibitors of either PI3K or Akt as well as by overexpression of the lipid phosphatase PTEN. In contrast, sTie2 shedding was enhanced by overexpression of either dominant negative PTEN, which increased Akt phosphorylation, or constitutively active, myristoylated Akt.

Conclusions— These findings demonstrate that VEGF regulates angiopoietin-Tie2 signaling by inducing proteolytic cleavage and shedding of Tie2 via a novel PI3K/Akt-dependent pathway. These results suggest a previously unrecognized mechanism by which VEGF may inhibit vascular stabilization to promote angiogenesis and vascular remodeling.

Tie2 plays an important role in vascular remodeling. We demonstrate that soluble Tie2 shedding is induced by vascular endothelial growth factor (VEGF) in a phosphoinositide 3-kinase/Akt-dependent manner. These findings suggest a novel mechanism by which VEGF may inhibit Tie2-mediated vascular stabilization to promote angiogenesis and vascular remodeling.

【关键词】  Tie VEGF phosphoinositide kinase Akt endothelium

Introduction

Tie2, a receptor tyrosine kinase (RTK) expressed predominantly on endothelial cells (ECs) and their embryonic precursors 1 is required for both embryonic and postnatal angiogenesis. 2 Substantial data now indicate that Tie2 and its ligands, the angiopoietins, regulate the transition between a mature stable vasculature and angiogenic or remodeling blood vessels. Angiopoietin-1 (Ang1) acts primarily as a Tie2 agonist to promote vessel maturation by inducing interactions between endothelial and periendothelial support cells, including pericytes and vascular smooth muscle cells. 3–5 In contrast, Angiopoieitin-2 (Ang2) is a context-dependent Tie2 antagonist, promoting vascular destabilization in part by opposing the effects of Ang1 6,7 but also through the activation of distinct endothelial signaling pathways. 8 Despite advances in understanding the effects of the angiopoietins on angiogenesis and vascular remodeling, little is known about the mechanisms regulating Tie2 expression and downregulation.

Previous reports have demonstrated that the extracellular domain of Tie2 is proteolytically cleaved, resulting in the release of a 75-kDa soluble Tie2 (sTie2) protein. Shedding of sTie2 from endothelial cells can be stimulated by phorbol myristate acetate (PMA), but otherwise nothing is known about the mechanisms regulating this process. 9 Soluble Tie2 is detectable in the serum of healthy individuals and is increased in a number of cardiovascular diseases, including congestive heart failure, coronary artery disease, and cancer. 10–12 Previous reports have demonstrated that Tie2 is expressed constitutively in the adult vasculature, 13 and its expression increases in vascular remodeling states, such as breast cancer. 14 Shedding of sTie2 in such conditions might regulate angiopoietin-Tie2 binding to facilitate angiogenesis. Alternatively, sTie2 shedding might simply be an indicator of normal receptor turnover. In either case, the function of sTie2 in endothelial cell biology remains unknown.

In this report, we investigated the role of soluble Tie2 in modulating Tie2 activity and cellular responses in vitro. sTie2 was found to bind both Ang1 and Ang2 and to inhibit ligand-mediated Tie2 phosphorylation and endothelial cell apoptosis. Interestingly, Tie2 shedding was both constitutive and induced by vascular endothelial growth factor (VEGF) via a phosphoinositide 3-kinase (PI3K)/Akt-dependent mechanism, which has not been described previously. This suggests a novel mechanism by which VEGF may control Tie2-Ang1 activity to downregulate the stabilizing effects of Ang1 and promote a proangiogenic state. These findings have implications for understanding the role of sTie2 in the regulation of vascular growth and remodeling.

Materials and Methods

Detailed descriptions of the methods, including generation of cell lines expressing Tie2, analysis of sTie2 concentration, protein binding assays, and generation and use of recombinant adenoviruses, are available in the supplemental materials (available online at http://atvb.ahajournals.org).

Antibodies and Reagents

Mouse monoclonal anti-Tie2 (clone 33), recognizing the extracellular domain of human and murine Tie2, has been described previously. 14 Mouse monoclonal antiphosphotyrosine (clone PY99) was from Santa Cruz Biotechnology. Mouse monoclonal anti-Ang1 and -Ang2 antibodies were from R&D Systems. Rat monoclonal anti-tubulin (clone YL1/2) was from Serotec. Rabbit polyclonal anti-Akt, anti-phosphoAkt (S473), and anticleaved caspase-3 antibodies were from Cell Signaling Technology. Ni 2+ -NTA agarose was from Qiagen. Phorbol-12-myristate 13-acetate (PMA), GM6001 (GM), bisindolylmaleimide I (BIS-I), PD98059 (PD), LY294002 (LY), SB203580 (SB), Isozyme-selective-Akt1/2 inhibitor VIII (AktVIIIi), 15 and Akt inhibitor X (AktXi) 16 were from Calbiochem. Recombinant Ang1, Ang2, and VEGF-A 165 were purchased from R&D Systems.

Results

Tie2 Is Constitutively Cleaved in Fibroblasts and HUVECs

Previous reports have demonstrated the presence of a soluble form of Tie2 (sTie2) in the cell culture medium of HUVECs and in human serum. To determine the mechanisms regulating sTie2 shedding, we first investigated whether sTie2 is produced in nonendothelial cells exogenously expressing Tie2. For the purposes of the present studies, cell culture conditioned medium (CM) is defined as serum-free medium in which cells are incubated for varying times and which contains secreted or shed proteins, such as sTie2. Cell lysates and CM from NIH 3T3 fibroblasts stably expressing murine Tie2 (3T3-mTie2) and HEK-293 cells stably expressing human Tie2 (293-hTie2) were analyzed along with lysates and media from HUVECs. Western blotting with an antibody against the Tie2 extracellular domain detected a 75-kDa protein in CM but not lysates from 3T3-mTie2 cells and HUVECs ( Figure 1 A). The relative molecular weight of sTie2 from 3T3-mTie2 cells and HUVECs is similar to that described previously. 9 Soluble Tie2 was undetectable in CM or lysates from 293-hTie2 cells by Western blotting ( Figure 1 A) or ELISA (data not shown), suggesting that the mechanisms of sTie2 shedding are conserved in cells of mesothelial origin. As expected, sTie2 was not detected from parental HEK-293 or NIH 3T3 cells ( Figure 1 B). To determine the time course of sTie2 shedding, conditioned media from HUVECs and 3T3-mTie2 cells were collected at varying times and sTie2 concentrations were quantified by ELISA. Tie2 shedding occurred rapidly in 3T3-mTie2 cells and peaked within 6 hours, whereas sTie2 concentrations in HUVEC conditioned media increased gradually over 48 hours ( Figure 1 C). In both cell types, peak sTie2 concentration was approximately 8 ng/mL at this time point.

Figure 1. sTie2 is shed from endothelial cells and fibroblasts in vitro. Detection of full-length Tie2 (150 kDa) or sTie2 (75 kDa) in conditioned media (CM) and cell lysates (Lys) from (A) 3T3-mTie2, 293-hTie2, and HUVECs or (B) parental 293 and 3T3 cells. C, Time course of sTie2 shedding from HUVECs and 3T3-mTie2 cells was determined by ELISA.

sTie2 Is Detectable In Vivo

To investigate Tie2 shedding in human serum, blood was collected from 22 healthy human volunteers, and serum was analyzed by ELISA to quantify the sTie2 concentration. sTie2 was detectable in human serum by both ELISA and immunoprecipitation (supplemental Figure 1 ). The mean concentration of sTie2 detectable by ELISA was 20.6 ng/mL and ranged from 5.2 to 37.0 ng/mL (supplemental Figure 1 ), which is consistent with the serum sTie2 concentrations observed by other investigators. 9,17 The variability in sTie2 concentration was also evident by immunoprecipitation and Western blotting (supplemental Figure 1).

sTie2 Binds Recombinant Ang1 and Ang2

Tie2 shedding both in vitro and in vivo suggested that it might have functional effects on Tie2 signaling via angiopoietin binding. To investigate this possibility, concentrated sTie2-containing CM from 3T3-mTie2 cells or control CM from parental NIH 3T3 cells was mixed with recombinant, 6-His-tagged Ang1 or Ang2. Ang1 or Ang2 was precipitated on nickel agarose, and sTie2 binding was analyzed by Western blotting. Binding of sTie2 was undetectable in the absence of either angiopoietin protein ( Figure 2 A, lane 6), but binding of sTie2 was readily detectable in the presence of either Ang1 or Ang2 ( Figure 2 A, lanes 7 and 8). As expected, no sTie2 binding was detectable when the angiopoietins were incubated with parental 3T3 conditioned media, which lack sTie2 ( Figure 2 A, lanes 2 and 3). These findings demonstrate that constitutively shed sTie2 can bind both Ang1 and Ang2 in vitro.

Figure 2. sTie2 binds Ang1 and Ang2 and inhibits ligand-mediated Tie2 phosphorylation. A, sTie2 from 3T3-mTie2 conditioned media (CM) binds recombinant 6-His-tagged Ang1 (A1) and Ang2 (A2) proteins. PBS without recombinant Ang protein was used as a negative control. Bound proteins were detected by Western blotting with the indicated antibodies. Recombinant Ang proteins (rA1, rA2) and 3T3-mTie2 CM (sTie2) served as controls for the migration of each protein. B and C, 293-hTie2 cells were treated with or without Ang1 (B) or Ang2 (C) (300 ng/mL) for 10 minutes in the presence of concentrated conditioned media from parental NIH 3T3 cells (–sTie2) or 3T3-mTie2 cells (+sTie2). Tie2 was immunoprecipitated (IP) from cell lysates, and proteins were Western blotted sequentially with antiphosphotyrosine and anti-Tie2.

sTie2 Inhibits Ang1- and Ang2-Mediated Tie2 Phosphorylation

To investigate the effects of sTie2 on ligand-mediated Tie2 activation, 3T3-mTie2 conditioned media was evaluated for its ability to inhibit Ang1- or Ang2-induced tyrosine phosphorylation of Tie2. In endothelial cells, Ang1 induces Tie2 phosphorylation. Previously, Ang2 had not been shown to effect significant Tie2 activation in endothelial cells, 6 although this has recently been described. 18 However, in nonendothelial cells both Ang1 and Ang2 reproducibly induce significant Tie2 activation. 6 Therefore, we tested whether sTie2 in 3T3-mTie2 conditioned media could inhibit angiopoietin-induced Tie2 phosphorylation in 293-hTie2 cells. 19 As expected, both Ang1 and Ang2 induced Tie2 phosphorylation in the presence of parental 3T3 conditioned media, which lacks sTie2 ( Figure 2B and 2 C). However, in the presence of sTie2-containing conditioned media, the ligand-induced increase in Tie2 phosphorylation was markedly attenuated ( Figure 2B and 2 C).

sTie2 Inhibits Ang1-Mediated Antiapoptotic Effects in HUVECs

To determine whether the inhibitory effects of sTie2 on Tie2 activation translate into effects on Tie2-mediated cellular responses, HUVECs were treated with Ang1 in the absence or presence of sTie2-containing conditioned media from 3T3-mTie2 cells, and effects on apoptosis were examined. 20 Apoptosis was induced in HUVECs with staurosporine, and effects of sTie2-containing CM from parental 3T3 cells or from 3T3 cells expressing mTie2 were tested. Treatment of serum-starved HUVECs with staurosporine for 90 minutes resulted in DNA fragmentation ( Figure 3 A) and caspase-3 cleavage ( Figure 3B and 3 C), consistent with induction of apoptosis, and these effects were inhibited by Ang1 in the presence of parental 3T3 conditioned media ( Figure 3A through 3 C). However, the antiapoptotic effects of Ang1 were blocked in the presence of sTie2-containing conditioned media from 3T3-mTie2 cells ( Figure 3A through 3 C). Notably, essentially identical results were observed when we used conditioned media from 3T3 cells expressing a kinase inactive mutant of Tie2, indicating that overexpression of active Tie2 in 3T3-mTie2 cells did not result in production of proteins that would adversely affect endothelial cell survival (data not shown). Taken together, these findings demonstrate that constitutively shed sTie2 binds both Ang1 and Ang2 and inhibits their effects on Tie2 activation and endothelial cell survival.

Figure 3. sTie2 inhibits Ang1-mediated endothelial cell survival. A, Serum-starved HUVECs were treated with or without Ang1 (300 ng/mL) in conditioned media from parental 3T3 cells (Control) or 3T3-mTie2 cells (sTie2). Effects on staurosporine-induced DNA fragmentation were quantified. * P <0.05 by ANOVA. B, HUVECs treated as in panel A were analyzed for apoptosis by Western blotting with anticleaved caspase-3 (arrow) or antitubulin as a loading control. C, Cleaved caspase-3 was quantified from Western blots from 3 separate experiments as in panel B and normalized to expression of tubulin. * P <0.05 by ANOVA.

sTie2 Shedding Is PMA- and VEGF-Inducible

Phorbol myristate acetate (PMA) has been shown to induce shedding of other RTKs, including Tie2, 9 and VEGF has been shown to induce shedding of the related Tie1 receptor in HUVECs. 21 To test the effect of VEGF on Tie2 shedding, HUVECs were stimulated with VEGF or with PMA as a positive control. Because primary endothelial cells undergo apoptosis after prolonged serum starvation, sTie2 shedding was examined after PMA significantly increased the amount of Tie2 shedding in HUVECs, as demonstrated by both ELISA and Western blotting ( Figure 4 A). Similarly, treatment of HUVECs with VEGF induced a significant increase in sTie2 concentration ( Figure 4 B).

Figure 4. sTie2 shedding is PMA- and VEGF-inducible, and constitutive sTie2 shedding is MMP-, p38 MAPK-, and PI3K/Akt-dependent. HUVECs were treated with or without PMA (100 nmol/L; A) or VEGF (20 ng/mL; B), and sTie2 was analyzed by ELISA and Western blotting (A). *** P <0.005; ** P <0.01. C, HUVECs were treated with GM6001 (GM6, 100 nmol/L), bisindolylmaleimide I (Bis-I, 100 nmol/L), or PD98059 (PD, 50 µmol/L), and sTie2 shedding was quantified by ELISA (*** P <0.005). D, HUVECs were treated with SB203580 (SB, 20 µmol/L) or LY294002 (LY, 50 µmol/L) alone or in combination, and sTie2 shedding was quantified (* P <0.05; ** P <0.01).

Constitutive sTie2 Shedding Is Matrix Metalloproteinase–, p38 MAPK-, and PI3K-Dependent

Our data indicated that Tie2 shedding occurs both constitutively and after ligand activation with VEGF. To investigate the mechanisms responsible for these 2 processes, we first used a panel of pharmacological inhibitors of various signaling pathways to explore the regulation of constitutive Tie2 shedding in HUVECs. Matrix metalloprotease (MMP) inhibition with the nonspecific inhibitor GM6001 significantly decreased sTie2 shedding from HUVECs, demonstrating that Tie2 cleavage is metalloprotease-dependent ( Figure 4 C). Shedding of other RTKs has been shown to be mediated by PKC and extracellular signal regulated kinase (ERK). Treatment of serum-starved HUVECs with either the nonselective PKC inhibitor bisindolylmaleimide I (Bis-I) or the MEK inhibitor PD98059 had no significant effect on constitutive Tie2 shedding ( Figure 4 C). Two other signaling pathways important for vascular growth and remodeling are the p38 MAP kinase pathway and the PI3K/Akt pathway. Inhibition of either p38 with SB203580 or PI3K with LY294002 significantly blocked Tie2 shedding into the conditioned media of HUVECs ( Figure 4 D). However, inhibition of both pathways simultaneously had no further effect on Tie2 shedding ( Figure 4 D), indicating that p38 and PI3K lie within a common pathway for the induction of Tie2 shedding. Taken together, these findings demonstrate that Tie2 shedding is MMP- and p38-dependent, as is the case for other RTKs. However, these data also demonstrate that Tie2 shedding is PI3K-dependent, indicating a novel role for the PI3K pathway in Tie2 shedding.

VEGF-Inducible sTie2 Shedding Is PI3K/Akt-Dependent

VEGF activates both p38 and PI3K, therefore we investigated whether VEGF-inducible Tie2 shedding was affected by inhibition of either of these pathways. HUVECs were treated with or without VEGF in the absence or presence of LY294002 or SB203580, and sTie2 release was measured by ELISA. Similar to our previous findings in unstimulated HUVECs, both the LY compound ( Figure 5 A) and the SB compound ( Figure 5 B) significantly inhibited VEGF-induced sTie2 release. As noted, p38 has been linked previously to RTK shedding but PI3K has not, therefore we focused our subsequent investigation on the role of the PI3K pathway in VEGF-mediated Tie2 shedding. To confirm the effects of PI3K on Tie2 shedding, we tested the effects of PTEN on this process, because PTEN is the major phosphatase in cells that hydrolyze the lipid products of PI3K. HUVECs were infected with recombinant adenoviruses to overexpress either wild-type (WT) catalytically inactive PTEN (C/S). PTEN-C/S acts as a dominant negative inhibitor in endothelial cells, 22 and it induced an increase in Akt phosphorylation ( Figure 5 C). The effects of PTEN were examined on both untreated and VEGF-treated cells. PTEN-WT significantly reduced Tie2 shedding in both untreated and VEGF-treated HUVECs compared with cells infected with control virus. Furthermore, dominant negative PTEN significantly increased Tie2 shedding both in the presence and absence of VEGF ( Figure 5 D). Taken together, these results demonstrate that both constitutive and VEGF-mediated Tie2 shedding are dependent on PI3K activity.

Figure 5. VEGF-inducible sTie2 shedding is PI3K/Akt-dependent. A, HUVECs were treated with VEGF (20 ng/mL) in the absence or presence of LY294002 (LY, 50 µmol/L), and sTie2 shedding was quantified by ELISA (* P <0.05). B, HUVECs were treated with VEGF (20 ng/mL) in the absence or presence of SB203580 (20 µmol/L) and conditioned media were analyzed as described in panel A (* P <0.05). C, HUVECs were infected with an empty adenovirus (EV) or adenoviruses encoding wild-type (WT) or dominant-negative PTEN (C/S), all at a multiplicity of infection of 100. Cell lysates were Western blotted with the indicated antibodies. D, HUVECs were uninfected (UN) or infected with the indicated adenoviruses, treated with or without VEGF (20 ng/mL) for 24 hour, and sTie2 concentration was determined in conditioned media (* P <0.05; *** P <0.005).

PI3K activates multiple downstream effector molecules, including Akt, which is essential for VEGF-induced angiogenesis and endothelial cell survival. 23 Therefore, we focused on the role of Akt in PI3K-dependent Tie2 receptor cleavage. HUVECs were first treated with or without VEGF in the presence or absence of a selective pharmacological inhibitor of Akt. The Akt inhibitor significantly decreased both basal and VEGF-induced sTie2 shedding ( Figure 6 A). Together with the results of PTEN inhibition, these findings suggested that Akt activation might be sufficient to induce sTie2 shedding. To investigate this possibility, HUVECs were infected with an adenovirus encoding constitutively active myristoylated Akt (myr-Akt) and sTie2 shedding was quantified. Compared with uninfected or control virus-infected cells, Admyr-Akt induced a dose-dependent increase in phospho-Akt ( Figure 6 B), which corresponded with significant increases in sTie2 shedding ( Figure 6 C). Additionally, the dominant-negative PTEN (PTEN C/S)-mediated increase in Tie2 cleavage was abrogated by pharmacological inhibition of Akt kinase activity ( Figure 6 D), confirming that PTEN-C/S-induced sTie2 shedding is mediated through Akt. Because Akt activation was sufficient to induce Tie2 shedding, we asked whether overexpression of myrAkt could induce this process in 293-hTie2 cells, in which sTie2 was undetectable ( Figure 1 A). Adenoviral infection of these replication-competent cells resulted in cell lysis and release of full-length Tie2 (supplemental Figure IIA and IIB), but AdmyrAkt failed to induce an increase in sTie2 shedding compared with control virus infection (supplemental Figure IIA and IIB). Taken together, these observations demonstrate that Akt activity is both necessary and sufficient for sTie2 shedding, but the necessary protease downstream of Akt is either not expressed in 293 cells or may not be activated by the same mechanisms in these cells.

Figure 6. Akt activation is necessary and sufficient for sTie2 shedding. A, HUVECs were pretreated with vehicle or an Akt inhibitor (Akt-VIIIi, 30 nM) then treated with VEGF (20 ng/mL) for 24 hours, and sTie2 shedding was quantified. B, HUVECs were uninfected (UN) or infected with a control, empty adenovirus (EV), or with the indicated multiplicity of infection (MOI) of an adenovirus encoding myristoylated Akt (myrAkt). Proteins were detected with antibodies against phospho-Akt (pAkt) or total Akt. C, HUVECs were infected with the indicated adenoviruses, and sTie2 concentration was quantified by ELISA 24 hours after changing cells to serum-free media. D, HUVECS were uninfected or infected with adenovirus encoding dominant-negative PTEN (C/S) and treated with vehicle or an Akt kinase inhibitor (Akt-Xi, 25 µmol/L), and sTie2 was quantified by ELISA (* P <0.05; ** P <0.01; *** P <0.005).

Discussion

Tie2 is an endothelial cell–specific RTK that is required for vascular growth and remodeling. Tie2 has been shown to be proteolytically cleaved, 9 yet the mechanisms and biological significance of this process have not been examined previously. Here, we demonstrate that Tie2 shedding occurs in both a constitutive and VEGF-inducible manner and that sTie2 is functional, as it binds both Ang1 and Ang2 to inhibit ligand-mediated receptor activation and downstream cellular responses. Further, we show that Tie2 shedding is regulated by PI3K/Akt- and p38 MAPK-dependent pathways and that Akt activation is both necessary and sufficient to induce sTie2 shedding. Although a role for p38 MAPK in RTK cleavage has been demonstrated previously, this is the first report to demonstrate a requisite role for the PI3K/Akt pathway in RTK shedding. Moreover, this is the first report to demonstrate a direct effect of VEGF on the regulation of the Tie2-Angiopioetin system via receptor cleavage ion of Tie2 membrane expression.

RTKs and other cell surface receptors are cleaved by metalloproteases, and the primary candidates for Tie2 shedding are the ADAM ( a d isintegrin a nd m etalloprotease) and ADAM-TS (ADAM with t hrombo s pondin motifs) families of metalloproteases. 24–26 ADAMs 10, 15, 17, and 19 have been implicated in cardiac development and angiogenesis 27–29 and are therefore potential candidates for the regulation of sTie2 shedding. The activation of RTK shedding by ADAMs has been linked to several signaling proteins, including ERK, p38 MAPK, and PKC. 24 In our studies, sTie2 shedding was mediated by p38 MAPK and PI3K/Akt, which regulated both basal (constitutive) and VEGF-inducible Tie2 shedding. Interestingly, the effect of simultaneous inhibition of p38 MAPK and PI3K on sTie2 shedding was not synergistic, suggesting that these proteins lie in the same pathway regulating Tie2 cleavage. Accordingly, evidence exists for crosstalk between the PI3K/Akt and p38 MAPK pathways. 30,31 Although p38 MAPK signaling has been linked to shedding of a variety of proteins, 32–34 to our knowledge this is the first report to demonstrate a role for PI3K/Akt in RTK shedding. Notably, VEGF-mediated activation of PI3K/Akt is required for endothelial cell survival and angiogenesis, 23 and our findings suggest that VEGF-mediated angiogenesis might involve sTie2 shedding. Moreover, our results provide potential functional relevance for this process, as it may serve to regulate the balance between angiogenesis and vascular quiescence.

Although the role of Tie2-Angiopoietin signaling in vascular growth and remodeling is quite complex, it is generally accepted that Ang1-mediated activation of Tie2 promotes vascular stabilization and quiescence, 4 whereas Ang2 acts in opposition to Ang1 to facilitate VEGF-mediated angiogenesis. 7 Our data demonstrate that sTie2 can bind both Ang1 and Ang2 and inhibit ligand-mediated Tie2 signaling. Because Ang1 and Ang2 have distinct context-dependent effects on the vasculature, the ultimate effect of sTie2 shedding on vascular growth or remodeling likely depends on the relative expression of the different Angiopoietin ligands within a given vascular bed. Tie2 has been shown to be activated by Ang1 in the quiescent adult vasculature, 13 and Ang1 inhibits VEGF-mediated increases in endothelial permeability. 5 In this context, VEGF-mediated Tie2 shedding would be predicted to result in vascular destabilization, thereby facilitating angiogenesis. Interestingly, the related Tie1 receptor, which has also been shown to play a role in vascular maturation and stabilization, is also proteolytically cleaved in both a VEGF- and PMA-inducible manner. 21,35 Taken together, these observations suggest that an important component of VEGF-mediated signaling is the activation of proteases that cleave and downregulate the Tie receptors, thereby shifting the vasculature from a quiescent to an angiogenic state.

In vivo, sTie2 has been detected in the serum of healthy human subjects, 9,36 a finding confirmed by our studies. In addition, recent reports have demonstrated increased levels of sTie2 in a variety of disease states characterized by vascular remodeling, including congestive heart failure, hypertension, and acute coronary syndromes. 11,12,37,38 Although the role of sTie2 is not entirely clear, serum sTie2 concentrations in patients with renal cell carcinoma correlated with disease stage and mortality, suggesting that sTie2 correlates with increases in tumor angiogenesis. 10

Pathophysiologically, it is also possible that enhanced sTie2 production serves as a marker of endothelial dysfunction, as chronic hyperactivation of multiple signaling pathways may result in enhanced Tie2 cleavage. Interestingly, preparations of HUVECs from different donors display significantly different basal levels of Tie2 shedding (data not shown), consistent with the wide range of serum concentrations of sTie2 detected in vivo. However, additional studies will be required to determine whether elevated sTie2 is a marker of aberrant signaling and cellular dysfunction (ie, a result of disease) or whether it contributes to the progression of vascular disease. The findings in this study provide a mechanistic basis from which to begin investigating these possibilities, as they provide key insights into a novel mechanism by which VEGF counteracts the vascular stabilizing effects of Tie2.

Acknowledgments

Sources of Funding

This work was supported in part by NIH grants R01HL70165 and R21DK069673 (to C.D.K.) and R36AG027584 (to C.M.F.); by a Grant-in-Aid (0655493U) from the Mid-Atlantic Affiliate of the American Heart Association (to C.D.K.); by grants from the British Heart Foundation and Medical Research Council of the United Kingdom (G0601295 and G0700288) (to A.A.); and by a Collaborative Wellcome Trust grant (063256/z/00/z, to A.A. and C.D.K.). C.M.F. was supported in part by a Fellowship Award from the UNCF-Merck Foundation and by the Medical Scientist Training Program at the Duke University School of Medicine.

Disclosures

None.

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作者单位：Department of Pharmacology and Cancer Biology (C.M.F., C.D.K.), Duke University Medical Center and the Duke University School of Medicine (C.M.F.), Durham, NC; the Department of Medicine (C.M.F., C.D.K.), Division of Cardiovascular Medicine, Duke University Medical Center, Durham, NC; and the Depart