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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2008;28:835.)
© 2008 American Heart Association, Inc.

Integrated Physiology/Experimental Medicine

Suppression of the Raf/MEK/ERK Signaling Cascade and Inhibition of Angiogenesis by the Carboxyl Terminus of Angiopoietin-Like Protein 4

Ying-Hua Yang; Yu Wang; Karen S.L. Lam; Ming-Hon Yau; Kenneth K.Y. Cheng; Jialiang Zhang; Weidong Zhu; Donghai Wu; Aimin Xu

From the Departments of Medicine (Y.-H.Y., K.S.L.L., M.-H.Y., K.K.Y.C., J.Z., W.Z., A.X.), Research Center of Heart, Brain, Hormone, and Healthy Aging (Y.-H.Y., Y.W., K.S.L.L., M.-H.Y., K.K.Y.C., J.Z., W.Z., A.X.), and Genome Research Center (Y.W.), and the Department of Pharmacology (A.X.), The University of Hong Kong, and the Guang Zhou Institute of Biomedicine and Health (D.W., A.X.), Chinese Academy of Sciences, China. Current affiliation for Y.-H.Y.: Department of Biology, Hai Nan Normal University, Hai Kou, China.

Correspondence to Aimin Xu, Department of Medicine, The University of Hong Kong, L8-40, New Laboratory Block, 21 Sassoon Road, Hong Kong. E-mail amxu{at}hkucc.hku.hk

	Abstract

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Objectives— Angiopoietin-like protein 4 (Angptl4) is a secreted glycoprotein that has recently been implicated in the regulation of angiogenesis and metastasis. This study aimed to investigate the structural and cellular basis underlying the biological actions of Angptl4.

Methods and Results— Circulating Angptl4 was proteolytically cleaved into NH2-terminal coiled-coil domain (N-Angptl4) and COOH-terminal fibrinogen-like domain (C-Angptl4). Using amino acid sequencing analysis, we identified a major cleavage site between Lys¹⁶⁸ and Leu¹⁶⁹ and a minor cleavage site between Lys¹⁷⁰ and Met¹⁷¹ in mouse Angptl4. C-Angptl4, but not N-Angptl4, potently inhibited both bFGF- and VEGF-induced cell proliferation, migration, and tubule formation in endothelial cells, and prevented neovascularization in mice. Treatment of C-Angptl4 with PNGase F (an N-glycosidase) ablated its N-linked glycosylation, and also significantly attenuated its antiangiogenic activities. C-Angptl4 blocked bFGF-induced activation of ERK1/2 MAP kinase, but had no obvious effect on Akt and P38 MAP kinase. Furthermore, C-Angptl4 abrogated bFGF-induced phosphorylation of Raf-1 and MEK1/2, whereas neither auto-phosphorylation of FGF receptor-1 nor activation of Ras was affected, suggesting that the blockage occurs at the level of Raf-1 activation.

Conclusions— The carboxyl terminus of Angptl4 alone is sufficient to suppress angiogenesis, possibly through inhibiting the Raf/MEK/ERK1/2 MAP kinase pathway in endothelial cells.

This study comprehensively investigated the structural and cellular basis underlying the antiangiogenic activities of angiopoietin-like protein-4 (Angptl4). We found that the carboxyl terminus of Angptl4 alone is sufficient to suppress neovascularization in an N-glycosylation–dependent manner. Furthermore, our results showed that Angplt4 inhibits the Raf-1/ERK1/2 MAPK pathway in endothelial cells.

Key Words: angiogenesis • angiopoietin-like proteins • glycosylation • MAP kinase • neovascularization

	Introduction

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Angiogenesis is the formation of new blood vessels from pre-existing primary plexus through the processes of vascular sprouting, branching, and differential growth to form more mature vascular networks.¹ Physiological angiogenesis is an essential process for reproduction, development, and wound repair. On the other hand, pathological angiogenesis play an important role in the disease progression such as cancer, diabetic retinopathy, atherosclerosis, and rheumatoid arthritis.²

Angiogenesis is the multiple step process orchestrated by a range of pro- and antiangiogenic factors, including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), thrombospondin, angiopoietins, and more recently, angiopoietin-like proteins (Angptl).^3,4 So far, 7 members of the Angptl family have been identified, all of which have a secondary structural organization similar to angiopoietins, including a NH₂-terminal coiled coil domain and COOH-terminal fibrinogen-like domain. However, unlike angiopoietins, none of the Angptl family members bind to the receptor tyrosine kinases Tie1 or Tie2.³

Angiopoietin-like protein 4 (Angptl4), also known as peroxisome proliferator-activated receptor (PPAR-) angiopoietin-related protein (PGAR), fasting-induced adipose factor (FIAF), or hepatic fibrinogen/angiopoietin-related protein (HFARP), is a circulating glycoprotein highly expressed in adipose tissue, liver, and placenta.^5–7 Angptl4 is cleaved in vitro and in vivo, and circulates in the blood stream mainly as truncated fragments.⁴ Recent studies from others and our laboratory have demonstrated Angptl4 to be an important regulator of glucose homeostasis, insulin sensitivity, and lipid metabolism.^8–11 In addition, growing evidence suggest that Angptl4 is a key player in angiogenesis. Although an earlier study suggested the potential proangiogenic activity of Angptl4,¹² more recent data from several independent laboratories have demonstrated Angptl4 as a potent antiangiogenic factor.^13–16 Expression of Angptl4 was found to be reduced in primary gastric cancer and several types of cancer cell lines, and was correlated with methylation of CpG islands in the 5' region of Angptl4.¹⁷ Using corneal neovascularization and Miles permeability assays, Ito et al showed that VEGF-induced angiogenesis and vascular leakiness were significantly inhibited by recombinant Angptl4.¹⁶ Transgenic mice that express Angptl4 in the skin showed remarkable suppression of tumor growth within the dermal layer associated with significantly decreased numbers of invading blood vessels.¹⁶ A more recent study by Galaup et al¹⁵ found that Angptl4 prevents tumor metastasis through inhibition of vascular permeability, tumor cell motility, and invasiveness.

In this study, we investigated the structural and cellular basis underlying the biological functions of Angptl4 in modulating angiogenesis. Using the Edman degradation-based amino acid sequencing method, we identified 2 endogenous proteolytic cleavage sites of Angptl4 within the linker region between its NH2-terminal coiled-coil domain and COOH-terminal fibrinogen-like domain. We found that the carboxyl terminus of Angptl4 was sufficient to suppress bFGF- and VEGF-induced endothelial cell proliferation, migration, tubule formation, and in vivo neovascularization in an N-glycosylation dependent manner. In addition, we elucidated the signaling pathways underlying the antiangiogenic activities of ANGPTL4.

	Methods

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For expanded methods and results, please see the supplemental materials, available online at http://atvb.ahajournals.org.

Affinity Purification of FLAG- and Myc-Tagged Proteins From HEK293 Cells
Cells were infected with recombinant adenoviruses encoding full-length Angptl4 or its various domains (50 pfu/cell) for 48 hours. The conditioned medium was collected, centrifuged to remove cell debris, and filtered through 0.22 µm filter. FLAG-tagged Angptl4 was purified using anti-FLAG M2 affinity gel as described previously.¹⁸ Myc-tagged NH2-terminus or COOH-terminus of Angptl4 was purified with the antic-Myc affinity agarose gel according to the manufacturer’s instruction (Sigma). Protein concentration was determined using the Bradford reagent (Pierce).

Identification of Protein Cleavage Sites by Amino Acid Sequencing
The purified protein was separated by SDS-PAGE, transferred to polyvinylidene difluoride membrane membrane, stained with Coomassie Brilliant Blue R-250, excised, and subjected to the Edman degradation amino acid sequencing analysis as we previously described.¹⁸

Cell Proliferation, Migration and Tubule Formation Assay, and In Vivo Neovascularization Studies
The methods were described in details in the supplementary information.

Statistics
Data are expressed as means±SD. Statistical analysis was conducted with the Student t test. The values of P<0.05 were considered statistically significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Angptl4 Is Cleaved at the Linker Region Between the Coiled-Coil Domain and Fibronogen-Like Domain
Previous studies from others and our laboratory have shown that both human and mouse Angptl4 are present in plasma as the cleaved forms.^6,9,19 To identify the precise cleavage site, we infected HEK293 cells with the recombinant adenoviruses encoding mouse full-length ANGPTL4. When Angptl4 was expressed in a serum-free condition, the majority of this protein detected in the extracellular medium was present as its full-length form with an apparent molecular weight of 65 kDa (Figure 1A). On the other hand, Angptl4 expressed in a serum-containing medium was cleaved into the truncated forms with the apparent mass of its carboxyl terminus at 47 kDa. The percentage of the cleaved form was progressively increased with the increases in serum concentrations. In the presence of 10% serum, the majority of Angptl4 in the extracellular media was cleaved, whereas its full-length form was virtually undetectable. In serum samples collected from C57 mice infused with recombinant adenoviruses encoding full-length Angptl4, the protein was also present exclusively as the cleaved forms (Figure 1B), suggesting that Angptl4 is cleaved both in vitro and in vivo in a serum-dependent manner. A similar serum-dependent cleavage pattern was also observed for human Angptl4 (data not shown).

View larger version (62K): [in this window] [in a new window]   Figure 1. Serum-dependent cleavage of Angptl4. A, Immunoblot for 25-µL of conditioned medium from HEK293 cells infected with adenovirus encoding FLAG-tagged Angptl4 (adv-Angptl4-F) or luciferase (adv-luc). B, Immunoblot for 1-µL of mouse serum collected at day 4 after viral injection. C, Schematic presentation of Angptl4 domain structure and its 2 cleavage sites.

Edman degradation-based amino acid sequencing analysis for the cleaved carboxyl terminus of mouse Angptl4 yielded a major NH₂-terminal sequence of LPKMTQL and a minor NH2-terminal sequence of MTQLIGL, which corresponded to the amino acid residues from 169 to 175 and from 172 to 178 respectively, suggesting that this protein was cleaved at 2 close sites: Lys¹⁶⁸ Leu¹⁶⁹ and Lys¹⁷¹ Met¹⁷² (Figure 1C). Amino acid sequence analysis for the cleaved human Angptl4 also identified a major NH₂-terminal sequence of LPEMAQP and a minor NH₂-terminal sequence of MAQPVDP (Figure 1C), indicating that the cleavage of human Angpl4 occurs at the same sites with its mouse homolog. Similar with Angptl3,²⁰ both cleavage sites of Angptl4 are located at the linker region between the coiled-coil domain and the fibrinogen-like domain.

C-Angptl4, but not N-Angptl4, Possesses the Antiangiogenic Properties
Several recent studies have demonstrated the role of Angptl4 as a novel modulator of angiogenesis,^13,14,16 although the structural basis underlying its actions remains largely elusive. Therefore, we produced full-length NH₂ terminus and COOH terminus of Angtpl4 from HEK293 cells infected with recombinant adenoviruses encoding N-Angptl4 and C-Angptl4, respectively (Figure 2A), and evaluated their effects in HUVECs. Full-length Angptl4 (F-Angptl4) was purified from serum-free conditioned medium. ³H-thymidine incorporation analysis showed that both F-Angptl4 and C-Angptl4 inhibited bFGF as well as VEGF-stimulated DNA synthesis in HUVECs in a dose-dependent manner (Figure 2B and 2C). The inhibitory activities of F-Angptl4 and C-Angptl4 were comparable. On the other hand, N-Angptl4 had no effect on cell proliferation.

View larger version (25K): [in this window] [in a new window]   Figure 2. F-Angptl4 and C-Angptl4, but not N-Angptl4, inhibits bFGF- and VEGF-induced HUVEC proliferation. A, SDS-PAGE analysis of recombinant F-Angptl4, C-Angptl4, and N-Angptl4. B and C, 3H-thymidine incorporation assay in HUVECs treated with bFGF (25 ng/mL), VEGF (10 ng/mL), or F-Angptl4, C-Angtpl4, or N-Angptl4 as indicated. *P<0.05; **P<0.01 (n=5 to 7).

We next performed a chemotaxis-type migration assay using a Boyden chamber to assess the effects of C-Angptl4 and N-Angptl4 on migration of HUVECs. This analysis showed that bFGF at 25 ng/mL significantly enhanced cell migration by 8-fold compared to those in untreated cells (Figure 3). C-Angptl4 but not N-Angptl4 inhibited bFGF-induced cell migration in a dose-dependent manner. Matrigel-based assay showed that bFGF-tubule formation was decreased by 42±3% and 74±6% after treatment with C-Angptl4 at the concentration of 1 µg/mL and 5 µg/mL, respectively (n=4 to 6, P<0.05). The inhibitory effects of C-Angptl4 on bFGF-induced migration and tubule formation were also observed in human microvascular endothelial cells (supplemental Figure I). Furthermore, C-Angptl4 inhibited VEGF-induced cell migration and tubule formation (data not shown).

View larger version (93K): [in this window] [in a new window]   Figure 3. C-Angptl4 inhibits cell migration and capillary tubule formation in HUVECs. A, Cells were seeded into the upper chamber of a Transwell, treated with bFGF or C-Angptl4 as indicated. Representative images of migrated HUVECs are shown. B, Capillary tubule formation of HUVECs grown in Martigel treated with bFGF or C-Angptl4.

To further confirm the antiangiogenic activity of Angptl4, we examined its effect on in vivo neovascularization using a Matrigel plug assay in C57 mice. 7 days after injection, Matrigel supplemented with bFGF was grossly red (supplemental Figure II), suggesting that it contained erythrocytes and hemoglobin. In contrast, Matrigel containing bFGF plus C-Angptl4 or F-Angptl4 was white, indicating the absence of erythrocytes and hemoglobin. In Matrigel supplemented with bFGF plus C-Angptl4 or F-Angptl4, the hemoglobin contents were significantly decreased compared to those treated with bFGF alone. Immunohistochemical staining also demonstrated that both F-Angptl4 and C-Angptl4 significantly decreased bFGF-induced new blood vessel formation. Direct visualization of vasculature by fluorescein isothiocyanate (FITC)-dextran showed a well-defined capillary network of blood vessels in plugs supplemented with bFGF alone, whereas this vasculature was disrupted after treatment with either F-Angptl4 or C-Angptl4 (supplemental Figure III). Quantitative analysis demonstrated a comparable activity of F-Angptl4 and C-Angptl4 in reducing vessel nodes, vessel ends, and vessel length induced by bFGF. On the other hand, N-Angptl4 had little effect. F-Angptl4 and C-Angptl4, but not N-Angptl4, attenuated VEGF-induced neovascularization in vivo (supplemental Figure IV). Furthermore, adenovirus-mediated overexpression of C-Angptl4, but not N-Angptl4 or luciferase control, markedly decreased the rates of mammary tumor growth and microvascular density in nude mice implanted with the estrogen receptor (ER) negative MDA-MB-231 cells (supplemental Figure V).

N-glycosylation of C-Angptl4 Contributes to Its Maximal Antiangiogenic Activities
Angptl4 has previously been shown to be posttranslationally modified by N-glycosylation.⁵ Indeed, the apparent molecular mass of C-Angptl4 was 47 kDa, which is 18 kDa larger than that predicted from its primary amino acid sequence (Figure 4A). Treatment of C-Angptl4 with PNGase-F (an N-glycosidase which cleaves all N-linked glycan chains), caused a significant reduction in its apparent molecular mass, whereas endoglycosidase-H (an enzyme that cleaves only high-mannose N-linked glycans) had no obvious effect. This data suggests that C-Angptl4 contains complex oligosaccharide structures. To investigate the potential role of N-glycosylation in modulating the antiangiogenic activities of C-Angptl4, we next produced the deglycosylated C-Angptl4 by digestion of the recombinant protein with PNGase-F, followed by affinity purification with sepharose beads coupled with anti Myc monoclonal antibody. Amino acid sequencing analysis demonstrated that C-Angptl4 had the same NH₂ terminus (MTQLIGL), suggesting that PNFase-F treatment did not cause the protein degradation. Compared to the glycosylated C-Angptl4, the ability of the deglycosylated C-Angptl4 to inhibit bFGF-induced cell proliferation was significantly decreased (Figure 4B). In addition, ablation of glycosylation attenuated the effects of C-Angptl4 in inhibiting bFGF-evoked cell migration and tubule formation in HUVECs (Figure 4C and 4D).

View larger version (36K): [in this window] [in a new window]   Figure 4. Depletion of N-glycosylation attenuates the antiangiogenic activity of C-Angptl4. A, SDS-PAGE analysis for recombinant C-Angptl4 digested with PNGase F or EndoH. B, C, and D show the effects of glycosylated and deglycosylated C-Angptl4 (dC-Angptl4, 5 µg/mL) on bFGF-induced DNA synthesis, cell migration, and tubule formation, respectively (n=6 to 8). *P<0.05; **P<0.01.

C-Angptl4 Attenuates bFGF-Induced Phosphorylation of ERK1/2 MAP Kinase, but not Akt and p38 MAP Kinase
Several key signaling cascades, including the ERK1/2 MAP kinase, Akt, and the P38 MAP kinase pathways, have been implicated in the modulation of both bFGF- and VEGF-induced angiogenic process.²¹ We next investigated whether or not the antiangiogenic effect of C-Angptl4 is mediated through inhibition of these signaling cascades. To this end, lysates from HUVECs under various treatments were probed with antiphosphospecific antibodies against the active forms of ERK1/2 (phospho-Thr²⁰²/Tyr²⁰⁴), Akt (phosphor-Ser⁴⁷³), and p38 (phosphor-Thr¹⁸⁰/Tyr¹⁸²). Treatment with bFGF induced a strong phosphorylation of the ERK1/2 MAP kinase in a time-dependent manner, with a maximal response being detected at 20 minutes (Figure 5). Cotreatment with C-Angptl4 markedly attenuated bFGF-induced phosophorylation of ERK1/2. Quantitative analysis showed that bFGF-evoked ERK1/2 phosphorylation was reduced by 46% in the presence of 1 µg/mL C-Angptl4 and almost completely abolished in the presence of 5 µg/mL C-Angptl4. Stimulation with bFGF also caused a time-dependent phosphorylation of Akt and P38 MAP kinase. However, these effects were not affected by C-Angptl4. C-Angptl4 also inhibited VEGF-induced phosphorylation of ERK1/2, but not VEGF-induced phosphorylation Akt and P38 MAP kinase (data not shown). Taken together, these results suggest that the antiangiogenic activities of C-angptl4 might be attributed to its specific suppression of the ERK1/2 MAP kinase pathway.

View larger version (46K): [in this window] [in a new window]   Figure 5. Effects of C-Angptl4 on bFGF-evoked phosphorylation of ERK1/2, Akt, and P38 MAP kinase. A, Equal amounts of cell lysates under various treatments were subjected to immunoblot analysis for phospho- and total ERK1/2, Akt, or P38. B, The quantitative data for samples from 20 minutes after bFGF stimulation **P<0.01 (n=5 to 6).

C-Angptl4 Impedes bFGF-Induced Activation of Raf-1 and MEK1/2, but not Autophosphorylation of FGF Receptor-1 and Activation of Ras
The MAPK kinase MEK1/2 is known to be the direct upstream kinase of the ERK1/2 MAP kinase.²² MEK1/2 is activated by its upstream kinase Raf-1 through phosphorylation at ser217/221. Raf-1 is a key relay point in the MAPK cascade, integrating positive and negative inputs from various upstream pathways.²³ We next investigated the effects of C-Angptl4 on these signaling molecules upstream of the ERK1/2 MAP kinase (supplemental Figure VI). As expected, bFGF stimulated phosphorylation of Raf-1 and MEK1/2 at Ser217/221. Both effects were markedly attenuated by C-Angptl4. Treatment with bFGF led to a significant increase of activated, GTP-bound Ras, an upstream activator of Raf-1. However, C-Angptl4 had no effect on bFGF-evoked activation of Ras or autophosphorylation of FGFR1, suggesting that attenuation of Raf-1 activation is the earliest target of C-Angptl4 in blocking the activation of the ERK1/2 MAP kinase signaling pathway (supplemental Figure VI). Notably, C-Angptl4–mediated suppression on bFGF-induced DNA synthesis, cell migration, and tubule formation was significantly reversed after transfection with the plasmid pCMV-RafCAAX which expressed constitutively active Raf-1 (supplemental Figure VII), further confirming that C-Angptl4 inhibits angiogenesis through suppression of the Raf1/MEK/ERK1/2 signaling cascade.

	Discussion

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In addition to its role in regulating lipid metabolism and insulin sensitivity, growing evidence suggests that Angptl4 is an important modulator of angiogenesis and vascular permeability. Two earlier reports suggested Angptl4 as a proangiogenic factor induced during arthritis and ischemia.^12,24 On the other hand, several recent independent studies demonstrated Angptl4 to be a potent inhibitor of angiogenesis and tumor metastasis in both cell culture system and animal models.^13–16 In this study, we showed that Angptl4 inhibits both bFGF- and VEGF-induced cell proliferation, migration, and tubule formation in vitro, and neovascularization in vivo, thus supporting the role of Angptl4 as an angiogenic inhibitor. Although we cannot explain the discrepancy between these findings, it is highly possible that the function of Angptl4 in modulating angiogenesis differs in different tissue contexts. Indeed, Angiopoietin-2 and Angptl1, the close relatives of Angptl4, have been reported to possess both anti- and proangiogenic activities in various experimental conditions.^25–28

The full-length Angptl4 undergoes a regulated proteolysis into 2 distinct truncated forms both in vitro and in vivo.^6,9,19 In this study, we have identified a major cleavage site at Lys¹⁶⁸ Leu¹⁶⁹ and a minor cleavage site at Lys¹⁷⁰ Met,¹⁷¹ both of which are located within the linker region between the coiled-coil domain and fibrinogen-like domain. We also observed a similar pattern of cleavage for human Angptl4. Notably, Angptl3, the closest member of Angptl4, is also cleaved within the linker region with a surrounding sequence context similar to Angptl4.²⁰ The NH₂-terminal coiled-coil domain of both Angptl3 and Angptl4 has been shown to be sufficient to induce hyperlipidemia by inhibiting the lipoprotein lipase activity.^10,20 On the other hand, the results from this study demonstrated that the carboxyl fibrinogen-like domain of Angptl4, but not its NH₂-terminal region, can act on endothelial cells to inhibit cell proliferation, migration, and tubule formation in vitro, and neovascularization in vivo. Notably, a more recent report demonstrated Angptl2 is also proteolytically cleaved during its secretion,²⁹ suggesting that proteolysis represents an important mechanism that regulates the multiple biological functions of the Angptl family.

In this study, we observed the N-glycosylation of Angptl4 at its COOH-terminal domain. Inspection of amino acid sequence of Angptl4 using the NetGlyc program identified a single predicted N-glycosyaltion site at Asparagine-177, which is conserved across several species of Angptl4 identified so far. Although the precise role of the N-linked glycan chains remains to be determined, our data suggest that this N-glycosylation is critically important for the antiangiogenic activity of C-Angptl4. Notably, Angptl4 has recently been shown to interact strongly with extracellular matrix of endothelial cells in a heparin/heparan sulfate proteoglycan–dependent manner.¹³ It is possible that N-linked glycan chains are involved in the interaction of Angptl4 with the heparins of excellular matrix or its unknown membrane receptors. It is also interesting to note that several endogenous inhibitors of angiogenesis, including endostatin,³⁰ thrombospondins,³¹ and angioarrestin,³² are also glycoproteins with the heparin-binding properties. Furthermore, a more recent study showed that posttranslational modifications, presumably glycosylation, are required for the ability of Angptl2 to stimulate expansion of hematopoitic stem cells.²⁹ These findings highlight the importance of glycosylation in modulating the biological functions of the Angptl family.

Although several members of the Angptl family have recently been implicated in regulating angiogenesis,⁴ the underlying mechanisms remain largely elusive. In this study, we showed that C-Angptl4 selectively impedes the activation of the ERK1/2 MPAK pathway, which is a central player in mediating both VEGF- and bFGF-induced angiogenesis.²¹ Many endogenous and pharmacological inhibitors of angiogenesis, such as platelet factor 4,³³ 16-kDa human prolactin,³⁴ the extracellular adherence protein from Staphylococcus aureus,³⁵ exert their functions by preferentially inhibiting the ERK1/2 MAPK signaling cascade. Within the ERK1/2 MAPK pathway, Raf-1 is an important relay point, integrating positive and negative inputs from upstream stimuli.²³ Our results clearly suggest Raf-1 as the earliest target responsible for C-Angpl4–mediated inhibition of the ERK1/2 MAPK pathway. Whether or not C-Angptl4 acts through its specific receptor(s) to transduce the negative signal onto Raf-1 needs to be clarified in the future study.

In summary, our present study provides novel evidence demonstrating that the COOH-terminal fibrinogen-like domain of Angptl4 alone is sufficient to inhibit angiogenesis possibly through impeding the ERK1/2 MAPK pathway at the level of Raf-1 inactivation. Further detailed elucidation of the receptor and postreceptor signaling events underlying the antiangiogenic properties could ultimately lead to the development of novel pharmacological agents for inhibiting pathological neovascularization as occurs in tumor growth and metastasis.

	Acknowledgments

 
Sources of Funding

This work was supported by the Hong Kong Research Grant Council (HKU 7609/05 M), the National 973 program of China (2006CB503908), and its matching funding and Outstanding Young Researcher Award from the University of Hong Kong (to A.X.).

Disclosures

None.

	Footnotes

 
Y.-H.Y and Y.W. contributed equally to this study.

Original received October 14, 2007; final version accepted February 27, 2008.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Folkman J. Angiogenesis: an organizing principle for drug discovery? Nat Rev Drug Discov. 2007; 6: 273–286.[CrossRef][Medline] [Order article via Infotrieve]

2. Pandya NM, Dhalla NS, Santani DD. Angiogenesis–a new target for future therapy. Vascul Pharmacol. 2006; 44: 265–274.[CrossRef][Medline] [Order article via Infotrieve]

3. Morisada T, Kubota Y, Urano T, Suda T, Oike Y. Angiopoietins and angiopoietin-like proteins in angiogenesis. Endothelium. 2006; 13: 71–79.[CrossRef][Medline] [Order article via Infotrieve]

4. Oike Y, Yasunaga K, Suda T. Angiopoietin-related/angiopoietin-like proteins regulate angiogenesis. Int J Hematol. 2004; 80: 21–28.[CrossRef][Medline] [Order article via Infotrieve]

5. Kim I, Kim HG, Kim H, Kim HH, Park SK, Uhm CS, Lee ZH, Koh GY. Hepatic expression, synthesis and secretion of a novel fibrinogen/angiopoietin-related protein that prevents endothelial-cell apoptosis. Biochem J. 2000; 346: 603–610.[CrossRef][Medline] [Order article via Infotrieve]

6. Mandard S, Zandbergen F, Tan NS, Escher P, Patsouris D, Koenig W, Kleemann R, Bakker A, Veenman F, Wahli W, Muller M, Kersten S. The direct peroxisome proliferator-activated receptor target fasting-induced adipose factor (FIAF/PGAR/ANGPTL4) is present in blood plasma as a truncated protein that is increased by fenofibrate treatment. J Biol Chem. 2004; 279: 34411–34420.[Abstract/Free Full Text]

7. Yoon JC, Chickering TW, Rosen ED, Dussault B, Qin Y, Soukas A, Friedman JM, Holmes WE, Spiegelman BM. Peroxisome proliferator-activated receptor gamma target gene encoding a novel angiopoietin-related protein associated with adipose differentiation. Mol Cell Biol. 2000; 20: 5343–5349.[Abstract/Free Full Text]

8. Ge H, Yang G, Yu X, Pourbahrami T, Li C. Oligomerization state-dependent hyperlipidemic effect of angiopoietin-like protein 4. J Lipid Res. 2004; 45: 2071–2079.[Abstract/Free Full Text]

9. Xu A, Lam MC, Chan KW, Wang Y, Zhang J, Hoo RL, Xu JY, Chen B, Chow WS, Tso AW, Lam KS. Angiopoietin-like protein 4 decreases blood glucose and improves glucose tolerance but induces hyperlipidemia and hepatic steatosis in mice. Proc Natl Acad Sci U S A. 2005; 102: 6086–6091.[Abstract/Free Full Text]

10. Sukonina V, Lookene A, Olivecrona T, Olivecrona G. Angiopoietin-like protein 4 converts lipoprotein lipase to inactive monomers and modulates lipase activity in adipose tissue. Proc Natl Acad Sci USA. 2006; 103: 17450–17455.[Abstract/Free Full Text]

11. Mandard S, Zandbergen F, van Straten E, Wahli W, Kuipers F, Muller M, Kersten S. The fasting-induced adipose factor/angiopoietin-like protein 4 is physically associated with lipoproteins and governs plasma lipid levels and adiposity. J Biol Chem. 2006; 281: 934–944.[Abstract/Free Full Text]

12. Le Jan S, Amy C, Cazes A, Monnot C, Lamande N, Favier J, Philippe J, Sibony M, Gasc JM, Corvol P, Germain S. Angiopoietin-like 4 is a proangiogenic factor produced during ischemia and in conventional renal cell carcinoma. Am J Pathol. 2003; 162: 1521–1528.[Abstract/Free Full Text]

13. Cazes A, Galaup A, Chomel C, Bignon M, Brechot N, Le Jan S, Weber H, Corvol P, Muller L, Germain S, Monnot C. Extracellular matrix-bound angiopoietin-like 4 inhibits endothelial cell adhesion, migration, and sprouting and alters actin cytoskeleton. Circ Res. 2006; 99: 1207–1215.[Abstract/Free Full Text]

14. Li KQ, Li WL, Peng SY, Shi XY, Tang HL, Liu YB. Anti-tumor effect of recombinant retroviral vector-mediated human ANGPTL4 gene transfection. Chin Med J (Engl). 2004; 117: 1364–1369.[Medline] [Order article via Infotrieve]

15. Galaup A, Cazes A, Le Jan S, Philippe J, Connault E, Le Coz E, Mekid H, Mir LM, Opolon P, Corvol P, Monnot C, Germain S. Angiopoietin-like 4 prevents metastasis through inhibition of vascular permeability and tumor cell motility and invasiveness. Proc Natl Acad Sci USA. 2006; 103: 18721–18726.[Abstract/Free Full Text]

16. Ito Y, Oike Y, Yasunaga K, Hamada K, Miyata K, Matsumoto S, Sugano S, Tanihara H, Masuho Y, Suda T. Inhibition of angiogenesis and vascular leakiness by angiopoietin-related protein 4. Cancer Res. 2003; 63: 6651–6657.[Abstract/Free Full Text]

17. Kaneda A, Kaminishi M, Yanagihara K, Sugimura T, Ushijima T. Identification of silencing of nine genes in human gastric cancers. Cancer Res. 2002; 62: 6645–6650.[Abstract/Free Full Text]

18. Wang Y, Xu A, Knight C, Xu LY, Cooper GJ. Hydroxylation and glycosylation of the four conserved lysine residues in the collagenous domain of adiponectin. Potential role in the modulation of its insulin-sensitizing activity. J Biol Chem. 2002; 277: 19521–19529.[Abstract/Free Full Text]

19. Ge H, Yang G, Huang L, Motola DL, Pourbahrami T, Li C. Oligomerization and regulated proteolytic processing of angiopoietin-like protein 4. J Biol Chem. 2004; 279: 2038–2045.[Abstract/Free Full Text]

20. Ono M, Shimizugawa T, Shimamura M, Yoshida K, Noji-Sakikawa C, Ando Y, Koishi R, Furukawa H. Protein region important for regulation of lipid metabolism in angiopoietin-like 3 (ANGPTL3): ANGPTL3 is cleaved and activated in vivo. J Biol Chem. 2003; 278: 41804–41809.[Abstract/Free Full Text]

21. Cross MJ, Claesson-Welsh L. FGF and VEGF function in angiogenesis: signalling pathways, biological responses and therapeutic inhibition. Trends Pharmacol Sci. 2001; 22: 201–207.[CrossRef][Medline] [Order article via Infotrieve]

22. Pearson G, Robinson F, Beers Gibson T, Xu BE, Karandikar M, Berman K, Cobb MH. Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions. Endocr Rev. 2001; 22: 153–183.[Abstract/Free Full Text]

23. Dhillon AS, Kolch W. Untying the regulation of the Raf-1 kinase. Arch Biochem Biophys. 2002; 404: 3–9.[CrossRef][Medline] [Order article via Infotrieve]

24. Hermann LM, Pinkerton M, Jennings K, Yang L, Grom A, Sowders D, Kersten S, Witte DP, Hirsch R, Thornton S. Angiopoietin-like-4 is a potential angiogenic mediator in arthritis. Clin Immunol. 2005; 115: 93–101.[CrossRef][Medline] [Order article via Infotrieve]

25. Daly C, Pasnikowski E, Burova E, Wong V, Aldrich TH, Griffiths J, Ioffe E, Daly TJ, Fandl JP, Papadopoulos N, McDonald DM, Thurston G, Yancopoulos GD, Rudge JS. Angiopoietin-2 functions as an autocrine protective factor in stressed endothelial cells. Proc Natl Acad Sci U S A. 2006; 103: 15491–15496.[Abstract/Free Full Text]

26. Gale NW, Thurston G, Hackett SF, Renard R, Wang Q, McClain J, Martin C, Witte C, Witte MH, Jackson D, Suri C, Campochiaro PA, Wiegand SJ, Yancopoulos GD. Angiopoietin-2 is required for postnatal angiogenesis and lymphatic patterning, and only the latter role is rescued by Angiopoietin-1. Dev Cell. 2002; 3: 411–423.[CrossRef][Medline] [Order article via Infotrieve]

27. Kubota Y, Oike Y, Satoh S, Tabata Y, Niikura Y, Morisada T, Akao M, Urano T, Ito Y, Miyamoto T, Nagai N, Koh GY, Watanabe S, Suda T. Cooperative interaction of Angiopoietin-like proteins 1 and 2 in zebrafish vascular development. Proc Natl Acad Sci USA. 2005; 102: 13502– 13507.[Abstract/Free Full Text]

28. Dhanabal M, LaRochelle WJ, Jeffers M, Herrmann J, Rastelli L, McDonald WF, Chillakuru RA, Yang M, Boldog FL, Padigaru M, McQueeney KD, Wu F, Minskoff SA, Shimkets RA, Lichenstein HS. Angioarrestin: an antiangiogenic protein with tumor-inhibiting properties. Cancer Res. 2002; 62: 3834–3841.[Abstract/Free Full Text]

29. Zhang CC, Kaba M, Ge G, Xie K, Tong W, Hug C, Lodish HF. Angiopoietin-like proteins stimulate ex vivo expansion of hematopoietic stem cells. Nat Med. 2006; 12: 240–245.[CrossRef][Medline] [Order article via Infotrieve]

30. John H, Radtke K, Standker L, Forssmann WG. Identification and characterization of novel endogenous proteolytic forms of the human angiogenesis inhibitors restin and endostatin. Biochim Biophys Acta. 2005; 1747: 161–170.[Medline] [Order article via Infotrieve]

31. Bocci G, Francia G, Man S, Lawler J, Kerbel RS. Thrombospondin 1, a mediator of the antiangiogenic effects of low-dose metronomic chemotherapy. Proc Natl Acad Sci U S A. 2003; 100: 12917–12922.[Abstract/Free Full Text]

32. Smagur A, Szary J, Szala S. Recombinant angioarrestin secreted from mouse melanoma cells inhibits growth of primary tumours. Acta Biochim Pol. 2005; 52: 875–879.[Medline] [Order article via Infotrieve]

33. Sulpice E, Bryckaert M, Lacour J, Contreres JO, Tobelem G. Platelet factor 4 inhibits FGF2-induced endothelial cell proliferation via the extracellular signal-regulated kinase pathway but not by the phosphatidylinositol 3-kinase pathway. Blood. 2002; 100: 3087–3094.[Abstract/Free Full Text]

34. Tabruyn SP, Nguyen NQ, Cornet AM, Martial JA, Struman I. The antiangiogenic factor, 16-kDa human prolactin, induces endothelial cell cycle arrest by acting at both the G0–G1 and the G2-M phases. Mol Endocrinol. 2005; 19: 1932–1942.[Abstract/Free Full Text]

35. Sobke AC, Selimovic D, Orlova V, Hassan M, Chavakis T, Athanasopoulos AN, Schubert U, Hussain M, Thiel G, Preissner KT, Herrmann M. The extracellular adherence protein from Staphylococcus aureus abrogates angiogenic responses of endothelial cells by blocking Ras activation. FASEB J. 2006; 20: 2621–2623.[Abstract/Free Full Text]

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