Published online before print February 7, 2008, doi: 10.1161/ATVBAHA.107.149674
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© 2008 American Heart Association, Inc.
Integrative Physiology/Experimental Medicine |
Angiopoietin-Related Growth Factor Enhances Blood Flow Via Activation of the ERK1/2-eNOS-NO Pathway in a Mouse Hind-Limb Ischemia Model
From the Laboratory of Vascular Biology and Metabolism (T.U., M.A., M.T., T.M., T.H., Y.O.), Center for Integrated Medical Research, and the Department of Cell Differentiation (T.U., Y.I., M.A., T.M., T.H., T.S., Y.O.), School of Medicine, Keio University, Tokyo, Japan; the Department of Ophthalmology and Visual Science (T.U., Y.I., H.T.), the Department of Microbiology (T.S., T.A.), and the Department of Molecular Genetics (T.U., K.M., M.T., M.Y., T.K., Y.O.), Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan; the Molecular Medicine Laboratories (K.Y.), Astellas Pharmaceutical Co Ltd, Tsukuba, Japan; the Department of Cardiology (R.S., T.M.), Nagoya University Graduate School of Medicine, Nagoya, Japan; and PRESTO (Y.O.), Japan Science Technology Agency (JST), Kawaguchi, Saitama, Japan.
Correspondence to Yuichi Oike, MD, PhD, Department of Molecular Genetics, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556, Japan. E-mail oike{at}gpo.kumomoto-u.ac.jp
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Abstract |
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Objective— Transgenic mice overexpressing angiopoietin-related growth factor (AGF) exhibit enhanced angiogenesis, suggesting that AGF may be a useful drug target in ischemic disease. Our goal was to determine whether AGF enhances blood flow in a mouse hind-limb ischemia model and to define molecular mechanisms underlying AGF signaling in endothelial cells.
Methods and Results— Intramuscular injection of adenovirus harboring AGF into the ischemic limb increased AGF production, which increased blood flow through induction of angiogenesis and arteriogenesis, thereby reducing the necessity for limb amputation. In vitro analysis showed that exposing human umbilical venous endothelial cells to AGF increased nitric oxide (NO) production through activation of an ERK1/2-endothelial NO synthetase (eNOS) signaling pathway. AGF-stimulated eNOS phosphorylation, NO production, and endothelial cell migration were all abolished by specific MEK1/2 inhibitors. Moreover, AGF did not restore blood flow to ischemic hind-limbs of either mice receiving NOS inhibitor L-NAME or eNOS knockout mice.
Conclusion— Activation of an ERK1/2-eNOS-NO pathway is a crucial signaling mechanism by which AGF increases blood flow through induction of angiogenesis and arteriogenesis. Further investigation of the regulation underlying AGF signaling pathway may contribute to develop a new clinical strategy for ischemic vascular diseases.
We sought to determine whether AGF enhances blood flow in a mouse hind-limb ischemia model and to define its molecular mechanisms. Activation of an ERK1/2-eNOS-NO pathway is crucial for AGF-induced angiogenesis and arteriogenesis. AGF signaling pathway may contribute to develop a new clinical strategy for ischemic vascular diseases.
Key Words: angiopoietin-related growth factor • therapeutic angiogenesis • nitric oxide • endothelial nitric oxide synthetase • extracellular signal-regulated kinase 1/2
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Introduction |
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Severe ischemic vascular diseases are often caused by atherosclerotic occlusion of arteries supplying blood to the myocardium and limbs. In such cases, bypass surgery or transluminal angioplasty is the most effective treatment available for restoring blood flow in the affected arteries. Recently, therapeutic angiogenesis mediated by angiogenic cytokines or bone marrow–derived mononuclear cells (BM-MNCs) has been proposed as a novel approach to treat patients with severe ischemic vascular disease who cannot undergo bypass surgery or angioplasty.1,2 Various angiogenic growth factors derived from implanted BM-MNCs have been shown to be key mediators of angiogenesis seen after implantation of BM-MNCs.3,4
We and others have independently identified several angiopoietin-like proteins (Angptls), including Angptl1/angiopoietin-related protein 1 (ARP1)/angioarrestin,5,6 Angptl2/ARP2,5,7 Angptl3,8,9 Angptl4/ARP4/PPAR
angiopoietin-related (PGAR)/fasting-induced adipose factor (FIAF)/hepatic fibrinogen/angiopoietin-related protein (HFARP),10–13 Angptl5,14 Angptl6/angiopoietin-like growth factor (AGF),15 and Angptl7.16 These Angptls are structurally similar to angiopoietins, which are characterized by a coiled-coil domain at the N terminus and a fibrinogen-like domain at the C terminus. However, whereas angiopoietins act via the Tie2 receptor tyrosine kinase, whose signaling regulates vascular stabilization and remodeling,17–20 Angptls do not bind to Tie2 or the related Tie1 receptor, suggesting that these orphan ligands function differently from angiopoietins. Angptls reportedly can function as either pro- or antiangiogenic factors14,20: Angptl1 and Angptl4 exert antiangiogenic effects,6,12 whereas Angptl2, Angptl3, and AGF act as proangiogenic factors.7,9,21
Hypoxia occurs in some tissues under both physiological and pathological circumstances. In both cases, angiogenesis is induced to maintain a sufficient oxygen supply, which is indicative of an important correlation between hypoxia and regulation of angiogenesis. Consistent with that relationship, expression of angiogenic mediators is commonly upregulated by hypoxia. AGF mRNA levels are upregulated by hypoxia,14,20,22 suggesting that AGF may play an important role during physiological or pathological angiogenesis. That most (>80%) AGF-deficient mice die by embryonic day 13 (E13), apparently as a result of vascular defects,23 is also consistent with this idea and suggests that AGF plays an important role during development of the vasculature.
Here, we used a mouse hind-limb ischemia model to investigate the efficacy of AGF in treating severe ischemia that would otherwise lead to necrosis and amputation of >80% of ischemic limbs. Our findings indicate that AGF increases blood flow in ischemic limbs through induction of both angiogenesis and arteriogenesis via an AGF-ERK1/2-eNOS-NO pathway. These findings indicate that further investigation of the regulation underlying AGF signaling pathway might contribute to develop a new clinical strategy for ischemic vascular diseases.
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Materials and Methods |
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Mice
BALB/c mice (8 to 12 weeks old) were purchased from CLEA (Tokyo, Japan), and eNOS knockout mice were purchased from The Jackson Laboratory (Bar Harbor, Me). All experimental procedures were performed under guidelines of Keio University and Kumamoto University for animal and rDNA experiments.
Hind-Limb Ischemia Model
After anesthetizing mice with pentobarbital (50 µg/g), the proximal part of the femoral artery and the distal portion of the saphenous artery were ligated and stripped, and all side branches were dissected free. At the time of surgery, either 5x109 plaque-forming units (pfu) of adenovirus harboring the mouse AGF gene (Ad-AGF),23 green fluorescent protein gene (Ad-GFP),23 or human VEGF165 gene (Ad-VEGF165)24 were injected into the ischemic muscle. Hind-limb blood flow was measured using a laser Doppler blood flow (LDBF) analyzer (Moor LDI; Moor Instruments). Blood flow-dependent changes in laser frequency were imaged using different colored pixels. After scanning, stored images were analyzed to quantify blood flow.
Histology
Isolated vastus and rectus femoris muscle tissues from ischemic limbs were washed with phosphate-buffered saline (PBS) and then fixed and prepared using the AMeX (Acetone Methylbenzoate Xylene) method.25 Each specimen was then cut into 8-µm sections, stained with anti-CD31 antibody (Pharmingen), photographed at 100x magnification (10 photographs per mouse) and analyzed using a blinded protocol. CD31-stained capillary endothelial cells were quantified as the number per single field; numbers of muscle fibers were determined as described previously.26
Angiography
On postoperative day 10, mice were anesthetized with sodium pentobarbital (50 µg/g), a 26-gauge catheter was inserted into the aorta via the left ventricle, and the cardiovascular system was perfused with 10 mL of PBS. For postmortem angiography, 3 mL of contrast medium were injected through the catheter, after which X-rays of the vasculature of the ischemic limb were obtained using a µFX-1000 system (Fujifilm). Images were analyzed using a BAS 5000 system (Fujifilm).
Cell Culture and Western Analyses
Human umbilical venous endothelial cells (HUVECs) were cultured as described.27 Cells were made quiescent by incubation for 8 hour in DMEM containing 0.1% BSA and then incubated in fresh medium containing 0.1% BSA and 1.0 µg/mL AGF for 0, 5, 15, 30, 60, 90, 120, or 240 minutes before collection for immunoblotting. Antibodies specific for phosphorylated ERK1/2Thr202/204, p38MAPKThr180/182, AMPKThr172, AktSer473, eNOSSer113, eNOSSer1177, eNOSThr495, and for the corresponding unphosphorylated proteins were obtained from Cell Signaling Technology. Proteins from the vastus and rectus femoris muscle tissues from ischemic mice limbs after injection of Ad-AGF or Ad-GFP were analyzed for immunoblotting. Antibody specific for AGF was used as described.15 Horseradish peroxidase-conjugated secondary antibodies were purchased from BIOSOURCE International.
Nitrite and Nitrate Measurement
HUVECs (1x105 cells) made quiescent by a 2-hour incubation in DMEM (Sigma) containing 0.1% BSA were treated with DMEM with or without 1.0 µg/mL AGF for 2 hours. In some cases, cells were pretreated for 60 minutes with a specific MEK1/2 inhibitor (U0126 at 30 µmol/L or PD980059 at 100 µmol/L; BIOMOL Research Laboratories Inc) before adding AGF. The cells were then centrifuged at 500g for 5 minutes at 4°C, and the supernatants were collected. Nitrite/nitrate levels, which reflect NO production, in supernatants were determined directly by high-performance liquid chromatography (HPLC) coupled to a Griess reagent-flow reactor.28
Cell Migration Assay
A migration assay was performed using Transwell polycarbonate membrane filters with 8.0-mm pore size (Costar), as described.12 EBM-2 containing 0.5% FBS with or without AGF (3.0 µg/mL) was loaded in the lower wells, and 3x104 HUVECs, which had been serum-starved for 24 hours and suspended in 100 mL of EBM-2 medium, were inoculated onto each upper well. After 4 hours incubation at 37°C, membranes were fixed with 100% methanol and stained with Giemsa solution. In some cases, cells were pretreated 60 minutes with a specific MEK1/2 inhibitor U0126 at 30 µmol/L or PD980059 at 100 µmol/L; BIOMOL Research Laboratories Inc), Akt inhibitor (LY294002 at 30 µmol/L; Calbiochem), or AMPK inhibitor (Compound C at 20 µmol/L; Calbiochem) before being inoculated onto each upper well. Cells on the upper surface of the membrane were removed with cotton swabs. The number of cells in untreated samples was represented as 100%. Each experiment was performed in triplicate and repeated 3 times.
Quantitative RT-PCR Analysis
Preparation of DNase-treated total RNA from the vastus and rectus femoris muscle tissues from ischemic mice limbs after Ad-AGF or Ad-GFP injection and reverse transcription were performed as described.21 PCR amplifications were performed using SYBR Premix Ex Taq Perfect Real Time kit (Takara Bio) in the Thermal Cycler Dice Real-Time System (Takara Bio). The cycle threshold value, determined using the second derivative, was used to calculate normalized expression of indicated genes relative to β-actin using Q-Gene software. The following primer pairs were used: β-actin, 5'-tggcacccagcacaatgaa-3' and 5'-ctaagtcatagtccgcctagaagca-3'; AGF, 5'-cttcaccaactggcagcactaca-3' and 5'-ctaggagtatcagcagcagctcgtggtc-3'; VEGF164, 5'- atgtgaatgcagaccaaagaaaga-3' and 5'-cgctctgaacaaggctcacag-3'; bFGF, 5'-ggacggctgctggcttctaa-3' and 5'-ccagttcgtttcagtgccacatac-3'; interleukin (IL)-1β, 5'-tccaggatgaggacatgagcac-3' and 5'-gaacgtcacacaccagcaggtta-3'; Angiopoietin1, 5'-tggaagtgttatcacccagttctca-3' and 5'-acaatgggctgttccaactcaag-3'.
Statistics
Data are presented as means±SD. Groups were compared using Student t test for per-comparison analysis. Data related to amputation frequency was evaluated using the log rank test. Values of P<0.05 were considered significant. N.S. indicates no significant difference between indicated 2 groups.
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AGF Enhances Blood Flow in Ischemic Mouse Hind-Limbs by Promoting Angiogenesis and Arteriogenesis
In a mouse hind-limb ischemia model, there is little spontaneous collateral vessel formation in response to induced ischemia; consequently, most ischemic hind-limbs become necrotic and must be amputated.29 To determine whether AGF can increase blood flow to ischemic limbs and thus reduce the number of amputations in this model, Ad-AGF was injected intramuscularly into the adductor muscle of the ischemic limb at the time of surgery. On postoperative day 4, Ad-AGF mice showed significantly greater expression of AGF mRNA and protein in hind-limb tissue than did controls (Figure 1A and 1B, n=5 in each group). In contrast, there was no difference in serum AGF levels (Figure 1C, n=5 in each group). The surgery led to severe occlusion of the right femoral artery, and there was no significant difference in the degree of postoperative ischemia between the 2 groups. On the other hand, blood flow in ischemic muscle gradually increased in mice receiving Ad-AGF, whereas severe ischemia persisted in controls receiving Ad-GFP (Figure 1D). Calculation of ischemic-to-nonischemic limb perfusion ratios 10 days after surgery revealed that Ad-AGF mice showed significant increases in blood flow, compared to controls (Figure 1E, n=8 in each group). Furthermore, very few mice administered Ad-AGF required amputation of the ischemic limb, whereas the ischemic limbs of most Ad-GFP control mice required amputation attributable to necrosis (Figure 1F, n=14 in each group, log rank test; P<0.01). We next compared the potency of AGF with that of VEGF165, which is one of most powerful proangiogenic factors. The effects of AGF on the increases of blood flow and limbs survival are not distinguishable compared to those of VEGF165 in mouse hind-limb ischemia model (Figure 1D through 1F, n=14 in Ad-VEGF165).
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To assess the extent of angiogenesis at the microcirculatory level, we determined capillary density by immunostaining tissue sections from ischemic tissues with the endothelial cell marker CD31 (Figure 2A). Quantitative analysis revealed that on postoperative day 10, capillary density was significantly greater in mice receiving Ad-AGF or Ad-VEGF165 than in those receiving Ad-GFP, whereas there was no significant difference in capillary density between Ad-AGF and Ad-VEGF165 groups (Figure 2B, n=8 in each group).
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We then undertook angiographic analysis to determine the extent to which collateral vessel formation contributed to enhanced blood flow. Figure 2C shows representative angiograms taken on postoperative day 10. Whereas numerous collateral vessels are evident in ischemic thigh muscle of a mouse treated with AGF or VEGF, only a few collateral vessels issuing from the internal iliac artery are seen in a control mouse. Thus AGF appears to increase blood flow in ischemic limbs by inducing both angiogenesis and arteriogenesis like VEGF.
AGF Stimulates Phosphorylation of ERK1/2 and eNOS and Enhances NO Production in Endothelial Cells
We next asked which signaling pathway is activated in endothelial cells when AGF exerts angiogenic effects. Transiently stimulation of HUVECs with recombinant AGF induced time-dependent increases in the level of ERK1/2 phosphorylation (left panels in Figure 3A and open columns in Figure 3B), suggesting that extracellular signal-regulated kinase (ERK) is an AGF target. Time-dependent phosphorylation of eNOS on Ser113 or Ser1177, but not on Thr495, were induced after ERK1/2 activation (left panels of Figure 3A and open columns of Figure 3C and 3D). Furthermore, pretreating cells with the specific MEK1/2 antagonist U0126 (30 µmol/L) for 60 minutes significantly inhibited AGF-induced phosphorylation of both ERK1/2 and eNOS (right panels in Figure 3A and closed columns in Figure 3B through 3D). Thus loss of ERK1/2 activation correlated with reduced eNOS phosphorylation. Because agonist-induced phosphorylation at any phosphorylatable residue is sufficient to activate eNOS, these findings suggest that AGF-mediated phosphorylation of ERK1/2 activates eNOS.
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Consistent with activation of eNOS, exposing HUVECs to AGF (1.0 µg/mL) significantly increased NO production, as evidenced by elevated levels of nitrite/nitrate in culture supernatants (supplemental Information I, available online at http://atvb.ahajournals.org). This AGF-mediated upregulation of NO production was significantly attenuated by pretreatment of cells with the specific MEK1/2 inhibitors, confirming that in endothelial cells an AGF-induced increase in NO production requires activation of the ERK1/2-eNOS pathway.
ERK1/2 Activation Is Required for AGF-Induced Endothelial Cell Migration
We previously reported that AGF promotes chemotactic but not mitotic activity in endothelial cells.21 Thus we asked whether ERK1/2 activation is required for AGF-induced endothelial cell migration. We found that AGF promoted migration of endothelial cells through a microchemotaxis membrane, and that this activity was significantly attenuated in cells pretreated for 60 minutes with U0126 (30 µmol/L) or PD980059 (100 µmol/L) (supplemental Information II). Thus ERK1/2 activation does appear to be required for AGF-induced endothelial cell migration.
NO Is Required for AGF-Induced Increases in Blood Flow Within Ischemic Hind-Limbs
To assess the role of NO production in AGF-induced improvement in blood flow, we analyzed AGF-induced restoration of blood flow in ischemic hind-limbs of model mice in the presence of the NOS inhibitor L-NAME. Wild-type mice administered Ad-AGF+PBS showed significant recovery of limb perfusion, whereas no recovery of blood flow was seen in mice treated with Ad-AGF+L-NAME or Ad-GFP+L-NAME (Figure 4A). Calculation of ischemic-to-nonischemic limb perfusion ratios 10 days after surgery revealed that blood flow in ischemic limbs of mice given Ad-AGF+L-NAME was significantly attenuated compared to mice receiving Ad-AGF+PBS (Figure 4B). There was also no difference in blood flow ratios seen in mice administered either Ad-AGF+L-NAME or Ad-GFP+L-NAME (Figure 4B), and most mice treated with Ad-AGF together with L-NAME lost the ischemic limb to necrosis, as did Ad-GFP+L-NAME controls (Figure 4C). Furthermore, angiography carried out on postoperative day 10 revealed that few collateral vessels were present in ischemic thigh muscles of mice treated with either AGF+L-NAME or GFP+L-NAME (Figure 4D). Loss of beneficial effects of exogenous AGF on ischemic limbs in the presence of L-NAME confirms that NO production is required for enhanced blood flow.
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To confirm the role of AGF-induced ERK1/2-NO signaling in ischemia-induced angiogenesis, we determined whether intramuscular injection of Ad-AGF into adductor muscle of the ischemic limb of eNOS knockout mice in a C57BL/6 background improved blood flow after surgery. In mice, angiogenic responses to ischemia in an ischemic hind-limb model differ in different genetic backgrounds29: C57BL/6 mice show well developed spontaneous collateral formation and decreased necessity for hind-limb amputation. We found that eNOS knockout mice in this background show significantly reduced blood flow ratios compared to control mice, although our data confirmed that in this background the necessity for ischemic-induced amputation was rare. Wild-type mice supplemented with Ad-AGF showed more rapid recovery of limb perfusion compared with Ad-GFP controls (data not shown). However, rapid recovery of blood flow ratio was abrogated in eNOS knockout mice (supplemental Information III). These data indicate that NO production is required to ameliorate blood flow mediated by Ad-AGF.
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Discussion |
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We previously showed that transgenic mice expressing AGF in epidermal keratinocytes (K14-AGF) exhibit increased numbers of microvessels in skin,21 suggesting that AGF promotes in vivo angiogenesis. We also demonstrated the in vivo proangiogenic activity of AGF in mouse Matrigel plug and corneal neovascularization assays, 2 independent indicators of de novo angiogenesis. Conversely, we found that blood flow was markedly reduced in the hind-limbs of AGF–/– mice compared to wild-type littermates.23 We therefore propose that AGF is an angiogenesis-inducing factor potentially useful to treat some ischemic diseases. As expected, the present study provides strong in vivo evidence that exogenous AGF effectively mediates therapeutic angiogenesis in a mouse hind-limb ischemia model. There was no significant difference in the degree of blood flow recovery and avoidance of limb amputation between the mice receiving Ad-AGF and Ad-VEGF165, suggesting the ability of AGF in therapeutic angiogenesis might be equal to those of VEGF. We found previously that AGF-induced vasculature in K14-AGF transgenic mice shows less vascular leakiness than that seen in K14-VEGF transgenic mice.15,21 Likewise, hemangioma formation observed in K14-VEGF transgenic mice is not seen in K14-AGF transgenic mice, suggesting that AGF could constitute a more favorable therapy.
In the present study, we show that AGF acts by increasing NO production via ERK1/2-eNOS signaling, which promotes both angiogenesis (de novo capillary vessel formation) and arteriogenesis (collateral vessel formation). Endothelium-derived NO (EDNO) plays important roles in regulating arteriogenesis and angiogenesis in the hind-limb ischemia model. EDNO production is induced by cytokines and growth factors, such as VEGF,30 bFGF,31 angiopoietin1,32 adiponectin,33 and netrin-1.34 Likewise, we found that phosphorylation-dependent eNOS activation and resultant EDNO production are critical for both AGF-mediated arteriogenesis and angiogenesis. In general, angiogenic factors activate cognate receptors expressed on endothelial cells, which in turn stimulate signal transduction kinases, promoting angiogenesis. For example, Akt, AMPK, and ERK1/2 all reportedly phosphorylate eNOS directly.34–37 We found that phosphorylation of ERK1/2, but not Akt or AMPK, was time-dependently increased when endothelial cells were stimulated with recombinant AGF protein. Specific ERK1/2 inhibitors effectively blocked AGF-induced eNOS phosphorylation and suppressed AGF-stimulated endothelial cell migration, indicating that proangiogenic effects of AGF-stimulated eNOS activation are attributable primarily to direct activation of ERK1/2 signaling. It was recently reported that ERK1/2 activation by netrin results in phosphorylation and activation of eNOS and production of NO, and that this in turn contributes to ERK1/2 activation and increased NO production through a positive feed-forward mechanism.34 A similar feed-forward cycle to augment NO production may occur when AGF promotes angiogenesis by stimulating the ERK1/2-eNOS-NO pathway. When we continuously stimulated endothelial cells with AGF after Ad-AGF transfection, not only ERK1/2 but also Akt and AMPK were phosphorylated (supplemental Information IV), whereas exogenous AGF activated ERK only. These findings suggest that Akt and AMPK signaling may be indirectly activated in cells constitutively expressing AGF. Because AMPK and Akt also reportedly phosphorylate eNOS and promote angiogenesis, we asked whether activation of AMPK and Akt signaling also contributes to AGF-induced angiogenesis using a migration assay. AGF-induced endothelial cell migration was significantly attenuated by pretreating cells with specific MEK1/2 inhibitors, but not with Akt and AMPK inhibitors (supplemental Information V). These findings indicate that activation of ERK1/2 signaling plays a pivotal role in AGF-induced angiogenesis.
Mice administered Ad-AGF showed significantly higher AGF protein level in hind-limb tissue than did Ad-GFP controls, whereas there was no difference between these 2 groups in serum AGF levels. This observation suggests that paracrine effects of AGF, but not systemic effects of AGF in circulation, on increased blood flow through angiogenesis and arteriogenesis is important in this mouse hind-limb ischemia model. BM-MNCs infiltrate wound sites and serve as sources of growth factors and cytokines that promote angiogenesis during wound healing. Several recent studies have shown that implantation of MNCs effectively promotes neovascularization of ischemic tissues, not only through incorporation of implanted progenitors into newly formed vessels, but also by serving as sources of secreted angiogenic factors.3,4 In that regard, these angiogenic factors induce neovascularization in a paracrine manner. We previously showed that AGF is broadly expressed in BM-MNCs,14,15 including cells of hematopoietic lineage, suggesting that AGF might be involved in BM-MNCs-induced neovascularization. Recently we showed that expression levels of IL-1β and bFGF but not VEGF and angiopoietin1 in ischemic skeletal muscle were significantly increased after Ad-AGF injection (supplemental Information VI). More recently others reported that implanted BM-MNCs also stimulate muscle cells to produce the angiogenic factor, IL-1β, thereby promoting neovascularization in ischemic tissues.38 AGF might be important as both a BM-MNC–secreted angiogenic factor and a skeletal muscle-stimulating factor to produce angiogenic factors, because AGF receptor in both endothelial cells and skeletal muscles are target tissues of AGF.23 Our finding that AGF enhances blood flow by inducing both angiogenesis and arteriogenesis suggests that AGF might function in mechanisms underlying the therapeutic effect of BM-MNC implantation in ischemic disease.
We recently reported that exogenous AGF antagonizes development of obesity and related insulin resistance.23 The microvasculature assists in heat dissipation at sites of active thermogenesis in peripheral tissues; therefore we hypothesized that in addition to its ability to directly increase energy expenditure, AGF may counteract obesity by stimulating thermogenesis, which would require development of microvasculature to dissipate heat in peripheral tissues.39 Thus our findings suggest that AGF could be key molecule for constituting an effective treatment for ischemic vascular disease and metabolic syndrome, which is associated with an increased risk of atherosclerotic vascular disease and subsequent myocardial or limb ischemia.
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Acknowledgments |
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We thank I. Ishimatsu and K. Fukushima for technical assistance with the experiments.
Sources of Funding
This work was supported by grants-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to Y.O.), and by research grants (to Y.O.) from the Astellas Foundation for Research on Metabolic Disorders, the Mitsui Life Social Welfare Foundation, the Japan Heart Foundation, the Takeda Science Foundation, and the Nateglinide Memorial Toyoshima Research and Education Fund.
Disclosures
None.
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Footnotes |
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Original received June 11, 2007; final version accepted January 16, 2008.
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References |
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