Angiopoietin-1 Induces Migration of Monocytes in a Tie-2 and Integrin-Independent Manner
Angiopoietin-1 (Ang-1) is an angiogenic growth factor that activates Tie-2 and integrins to promote vessel wall remodeling. The recent finding of the potential proatherogenic effects of Ang-1 prompted us to investigate whether Ang-1 promotes monocyte chemotaxis, endothelial binding, and transendothelial migration, key events in the progression of atherosclerosis. Here, we show that Ang-1 induces chemotaxis of monocytes in a manner that is independent of Tie-2 and integrin binding but dependent on phosphoinositide 3-kinase and heparin. In addition, Ang-1 promoted phosphoinositide 3-kinase-dependent binding of monocytes to endothelial monolayers and stimulated transendothelial migration. Fluorescence-activated cell sorting analysis showed that exogenous Ang-1 adheres directly to monocytes as well as to human umbilical endothelial cells, but neither Tie-2 mRNA nor protein were expressed by primary monocytes. Although Ang-1 binding to human umbilical endothelial cells was partially Tie-2 and integrin dependent, Ang-1 binding to monocytes was independent of these factors. Finally, preincubation of monocytes with soluble heparin abrogated Ang-1 binding to monocytes and migration, and partially prevented Ang-1 binding to human umbilical endothelial cells. In summary, Ang-1 induces chemotaxis of monocytes by a mechanism that is dependent on phosphoinositide 3-kinase and heparin but independent of Tie-2 and integrins. The ability of Ang-1 to recruit monocytes suggests it may play a role in inflammatory angiogenesis and may promote atherosclerosis.
Angiopoietins (Angs) are a family of polypeptide ligands that bind to Tie-2, an endothelial cell-specific receptor tyrosine kinase that is required for vascular remodeling, stabilization, and mural cell recruitment.1 Ang-1 and Ang-2 are unique in that they elicit distinct responses from the same Tie-2 receptor.1 Ang-1 can also bind to and elicit functions via integrins and extracellular matrix proteins, such as vitronectin.2 Transgenic overexpression of Ang-1 in the skin or systemic delivery of Ang-1 dramatically blocks increases in vascular permeability in response to vascular endothelial growth factor3 and reduces microvascular leakage in inflammatory disease.4 Ang-1 also inhibits vascular endothelial growth factor-mediated leukocyte adhesion to endothelial cells by preventing upregulation of cell surface adhesion molecules.5,6 These studies led to the view that Ang-1 is a vascular protective agent.
The function of Ang-1 in the context of vascular disease is more complex than originally contemplated. First, it has been reported that postintracoronary perfusion of the allografts with an adenovirus encoding human Ang-1 can promote the development of cardiac allograft arteriosclerosis in rats.7 Second, Ang-1 gene expression was strongly correlated with both femoral and carotid-radial artery pulse-wave velocity waveforms in the peripheral blood monocytes of hypertensive patients, indicating a link between arterial stiffness and hypertensives.8 Arterial stiffness is an important independent predictor of cardiovascular mortality in hypertensive patients, and pulse-wave velocity is a useful index of arterial stiffness and an independent marker of cardiovascular adverse outcome in hypertensives.9 Finally, eosinophils,10 neutrophils,11,12 and a subset of monocytes that contribute to tumor angiogenesis13 were reported to express Tie-2. In addition, Ang-1 was shown to induce migration of eosinophils10 and neutrophils,11 and this is highly relevant because leukocyte migration and invasion into the arterial wall is critical for the development of atherosclerotic lesions and in the development of a vulnerable plaque.14
In this study, we investigated the expression of Tie-2 on human peripheral blood monocytes and monocyte-derived cell lines and their migratory responses to Ang-1 stimulation. We show for the first time that Ang-1 binds to monocytes and induces migration of these cells via a mechanism that is independent of Tie-2 and integrins.
Full methods are described in the supplemental data, available online at http://hyper.ahajournals.org.
Ang-1 Induces Phosphoinositide 3-Kinase (PI3K)-Dependent Monocyte Migration
Ang-1 induced a concentration-dependent increase in migration of monocytes (Figure 1A), whereas Ang-2 had no effect. Migration could be abrogated by prior heat-inactivation of the ligand (Figure 1B). Blockade of Ang-1 through preincubation with recombinant soluble Tie-2 receptor (rTie-2-Fc) also prevented migration, indicating the ligand-specific nature of the response (Figure 1C). Checkerboard analysis demonstrated that Ang-1 is capable of promoting both chemotaxis and chemokinesis of monocytes (Table).
Many of the functions attributed to Ang-1 are mediated by signaling via the PI3K pathway,15,16 and monocyte chemotaxis is reported to be PI3K dependent.17 The PI3K inhibitor, LY29004, abrogated Ang-1–induced monocyte migration (Figure 1D), whereas the MEK-1 inhibitor, PD98059, did not prevent Ang-1–mediated monocyte migration (data not shown), suggesting that the PI3K pathway is required for this response.
Monocytes, on activation, adhere firmly to the endothelium and transmigrate through the endothelial cell monolayer. To examine the role of Ang-1 in monocyte transmigration, human umbilical endothelial cells (HUVECs) were pretreated with tumor necrosis factor (TNF)-α, and monocytes were allowed to migrate across the endothelial monolayer toward Ang-1 (400 ng/mL) for 2 hours. Ang-1 induced a significant increase in monocyte transendothelial migration as compared with control treated cells (Figure 1E), suggesting that Ang-1 can induce direct binding and migration of monocytes.
Ang-1 Induces PI3K-Dependent Monocyte Adhesion
During inflammation, monocytes are recruited to sites of endothelial cell injury and roll along the vascular endothelium, where they become activated. We assessed whether treatment of endothelial cells with Ang-1 or Ang-2 could stimulate adhesion of monocytes. Both Ang-1 (200 ng/mL) and Ang-2 (200 ng/mL) induced a significant increase monocyte adhesion after 1 hour of coculture (Figure 2A and 2B). TNF-α at 10 ng/mL was used as a positive control. Inhibition of Ang-1 or Ang-2 with rTie-2-Fc produced a significant decrease in monocyte adhesion (Figure 2C). Moreover, inhibition of endothelial Tie-2 receptor using Tie-2 inhibitory peptide (NLLMAAS) also led to a decrease in Ang-1–induced monocyte adhesion (Figure 2D). To further elucidate the signaling pathway, inhibition of the PI3K pathway with LY294002 blocked Ang-1–mediated monocyte adhesion. In contrast, heparin pretreatment had no effect on Ang-1– or Ang-2–induced adhesion, indicating a heparin-independent but PI3K-dependent monocyte adhesion event.
Ang-1 Binds Directly to the Monocyte Cell Surface
HUVECs, primary monocytes, and the monocyte-derived cell lines, U937 and THP-1, express endogenous cell surface Ang-1 (data not shown). Thus, to differentiate between endogenous and exogenously administered Ang-1, a recombinant human Ang-1 was used as the source of Ang-1 (Ang-1–His). Binding of Ang-1–His was detected on THP-1, U937 cells, and HUVECs (Figure 3A through 3C), whereas binding of Ang-2 was only detectable on HUVECs (Figure 3C).
Ang-1–Induced Monocyte Migration Independent of Tie-2
To assess the involvement of Tie-2 in the Ang-1–mediated monocyte migratory response, monocytes were preincubated with the inhibitory Tie-2 peptide or a blocking Tie-2 antibody. Inhibition of Tie-2 did not affect Ang-1–induced monocyte migration (Figure 4A), whereas Tie-2 inhibitory peptide significantly abrogated Ang-1–induced HUVEC migration (Figure 4B), suggesting that the Ang-1–induced monocyte response is a Tie-2-independent function. Moreover, the Tie-2 blocking peptide had no effect on the direct binding of Ang-1 to monocytes (Figure 4C), whereas it partially abrogated Ang-1 binding to HUVECs, which express Tie-2 (Figure 4D). RT-PCR (Figure 4E), Western blot analysis (Figure 4F), and ELISA (Figure 4G) demonstrated the absence of Tie-2 mRNA and protein in monocytes and monocyte-derived cell lines. Thus, Ang-1 induces monocyte migration in a Tie-2-independent manner.
Ang-1–Induced Monocyte Migration Is Integrin Independent
Ang-1 has been shown to mediate cellular functions in endothelial and nonendothelial cells via direct binding and interaction with integrins.2,18,19 Preincubation of monocytes with RGD-based peptides, which inhibit integrin binding, did not prevent Ang-1–induced monocyte migration (Figure 5A) but blocked Ang-1–induced HUVEC migration (Figure 5B). In addition, pretreatment of cells with EDTA, a pan-integrin inhibitor, also did not affect Ang-1 binding to THP-1 cells (Figure 5C), and only partially prevented Ang-1 binding to HUVECs (Figure 5D). The results show that integrins play no role in Ang-1–induced monocyte chemotaxis.
Ang-1–Induced Monocyte Migration Is Heparin Dependent
Incubation of either primary human monocytes or HUVECs with soluble heparin prior to the assay abrogated Ang-1–induced migration of both cell types (Figure 6A and 6B). In addition, incubation of THP-1 or U937 with soluble heparin prior to Ang-1–His administration prevented Ang-1 binding (Figure 6C and 6D), suggesting a heparin dependence of this function in these cell types. Incubation of HUVECs with heparin partially inhibited Ang-1 binding, but heparin had no effect on the binding of Ang-2 to HUVECs (Figure 6E). Thus, the binding of Ang-1 to monocytes is via a heparin-dependent mechanism only, whereas Ang-1 binding to endothelial cells can be accounted for by Tie-2, integrin, and heparin-dependent mechanisms.
The transmigration of monocytes into the subendothelial space in response to endothelial injury plays a significant role in the development of early atherosclerotic lesions, as well as augmenting progression of the disease in later stages.20 The finding that Ang-1 failed to protect against arteriosclerotic lesion formation7 prompted us to examine the effects of Ang-1 on monocyte migration in vitro. We observed that Ang-1, but not Ang-2, induced a dose-dependent migration of human primary monocytes and monocytic cell lines, which could be inhibited by preincubation of Ang-1 with recombinant soluble Tie-2. Additionally, Ang-1 also induced a significant increase in monocyte transmigration. However, preincubation of the cells with a blocking Tie-2 antibody or an inhibitory Tie-2 peptide had no effect on the Ang-1–induced migration of monocytes or Ang-1 binding to these cells but did partially inhibit Ang-1 binding to HUVECs. Demonstrating that the observed Ang-1 effects on monocytes are not mediated via Tie-2.
Tie-2 expression has been demonstrated on eosinophils10 and neutrophils,11 and Ang-1 has been shown to induce Tie-2-dependent migration of these cells.11 Tie-2 expression was also observed on a subset of monocytes, designated Tie-2-expressing monocytes, that contribute to tumor angiogenesis13 and on the murine macrophage RAW 264.7 cell line by immunofluorescent staining.21 We undertook RT-PCR, Western blot analysis, and fluorescence-activated cell sorter (FACS) for Tie-2, but did not detect it in primary monocytes or monocytic cell lines. Functional assays assessing the migratory capacity of monocytes in response to Tie-2 inhibition showed that neither the anti-Tie-2 antibody nor the Tie-2 inhibitory peptide could block Ang-1–induced monocyte migration. These findings led us to conclude that Tie-2 is not involved in the observed Ang-1–dependent migration of monocytes. However, pretreatment of Ang-1 with recombinant soluble Tie-2 protein, particularly at higher concentrations, markedly inhibited Ang-1–induced monocyte migration in a concentration-dependant manner, confirming the effect to be Ang-1 specific.
Several studies have shown that the functional effects of Ang-1 can be achieved without ligation of the Tie-2 receptor. Signaling through integrins is one such alternative mechanism. Ang-1 has been shown to induce adhesion and spreading of endothelial cells via α5β1 and α5β5 integrins, and Ang-1 adhesion in Tie-2-negative fibroblasts was mediated by these integrins.2 Ang-1 can also induce integrin-dependent survival of Tie-2-negative cardiac and skeletal myocytes,18 and recently, α5β1 integrin was shown to directly and stably interact with the Tie-2 receptor itself to promote Ang-1–dependent angiogenesis.19 In our current study, integrin inhibitory peptides were used to determine whether integrins were involved in the monocyte response to Ang-1. These peptides did not affect migration, suggesting that integrins are not mediating this response. FACS analysis showed that Ang-1 binding was unaffected by pan-integrin inhibitor EDTA, suggesting that Ang-1 fails to interact with monocyte integrins.
More recently, low-shear flow conditions were identified to increase the expression of Ang-2 in human endothelial cells,22 and Ang-2 potentiates TNF-α-induced monocyte adhesion.23 In FACS-based binding, we show that Ang-1, but not Ang-2, binds to monocytes, whereas both Ang-1 and Ang-2 bind to HUVECs. Ang-1 binding was unaffected by the preincubation of the cells with Tie-2 peptide or the pan-integrin inhibitor EDTA. Soluble heparin abrogated the binding of Ang-1 to monocytes but only partly inhibited the binding to HUVECs. Preincubation of HUVECs with Tie-2 inhibitory peptide, EDTA, and heparin abrogated the binding of Ang-1, indicating that Ang-1 binding to HUVECs is mediated by 3 separate entities. Future work will attempt to identify the heparin-dependent receptor by proteomic analysis.
Activation of the PI3K pathway has been shown to be essential for transendothelial monocyte migration and as a consequence is implicated in chronic inflammatory diseases, such as atherosclerosis.24 Ang-1 promotes endothelial cell survival via activation of the PI3K signal transduction pathway. Pharmacological inhibition of the PI3K pathway inhibited monocyte migration, demonstrating for the first time a function that is Ang-1 and PI3K dependent but not mediated via Tie-2. However, the precise cell surface structures ligated by Ang-1 to activate the PI3K signal transduction pathway, to promote monocyte chemotaxis, remain unknown.
Ang-1 is an angiogenic growth factor that activates Tie-2 and integrins to promote vessel wall remodeling. This study demonstrates, for the first time, that Ang-1 binds to monocytes and induces migration of these cells via a mechanism that is independent of Tie-2 and integrins, but requires activation of PI3K pathway. In addition, it shows that the monocyte binding and migration is abrogated by soluble heparin and that Ang-1 binding to endothelial cells is also partially heparin dependent. Collectively, these findings indicate that Ang-1 can function as a proinflammatory mediator. High levels of Ang-1 may contribute to vascular/inflammatory disorders, such as atherosclerosis, and we advise caution when considering using Ang-1 as a therapeutic agent to tackle compromised vascular status in individuals with cardiovascular disease.
Sources of Funding
This work was supported by grants from the Medical Research Council (G0601295 and G0700288) and British Heart Foundation (RG/09/001/25940, PG/06/114 and BHF CoRe).
S.A. and M.J.C. contributed equally to this work.
- Received April 27, 2010.
- Revision received May 14, 2010.
- Accepted July 12, 2010.
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