Published online before print July 8, 2004, doi: 10.1161/01.RES.0000138018.61065.d1
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© 2004 American Heart Association, Inc.
Integrative Physiology |
Angiogenic Effects of Adrenomedullin in Ischemia and Tumor Growth
From the From the Department of Cardiovascular Medicine (S.I., T.S., T.A., P.N., N.T., H.I., Y.Z., A.E., R.N.), Graduate School of Medicine, University of Tokyo; and Department of Experimental Nursing (N.M.), Faculty of Nursing, Fukuoka Prefectural University, Japan.
Correspondence to Takayuki Shindo, MD, PhD, Department of Cardiovascular Medicine, University of Tokyo Graduate School of Medicine, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. E-mail shindo-tky{at}umin.ac.jp
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Abstract |
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Adrenomedullin (AM) is a novel vasodilating peptide involved in the regulation of circulatory homeostasis and implicated in the pathophysiology of cardiovascular disease. We tested the hypothesis that AM also possesses angiogenic properties. Using laser Doppler perfusion imaging, we found that AM stimulated recovery of blood flow to the affected limb in the mouse hind-limb ischemia model. AM exerted this effect in part by promoting expression of vascular endothelial growth factor (VEGF) in the ischemic limb, and immunostaining for CD31 showed the enhanced flow to reflect increased collateral capillary density. By enhancing tumor angiogenesis, AM also promoted the growth of subcutaneously transplanted sarcoma 180 tumor cells. However, heterozygotic AM knockout mice (AM+/–) showed significantly less blood flow recovery with less collateral capillary development and VEGF expression than their wild-type littermates. Similarly, mice treated with AM22-52, a competitive inhibitor of AM, showed reduced capillary development, and growth of sarcoma 180 tumors was inhibited in AM+/– and AM22-52–treated mice. Notably, administration of VEGF or AM rescued blood flow recovery and capillary formation in AM+/– and AM22-52–treated mice. In cocultures of endothelial cells and fibroblasts, AM enhanced VEGF-induced capillary formation, whereas in cultures of endothelial cells AM enhanced VEGF-induced Akt activation. These results show that AM possesses novel angiogenic properties mediated by its ability to enhance VEGF expression and Akt activity. This may make AM a useful therapeutic tool for relieving ischemia; conversely, inhibitors of AM could be useful for clinical management of tumor growth.
Key Words: angiogenesis • ischemia • vascular biology • vascular endothelial growth factor
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Adrenomedullin (AM) is a vasodilating peptide first identified in human pheochromocytoma based on its ability to increase cAMP levels in platelets.1 Since then, AM has been shown to be synthesized by a variety of tissues and cell types, and high levels of AM have been found in the adrenal medulla, heart, lung, and kidney.2 AM also has been found circulating in plasma3 after secretion mainly by vascular cells.4 Apart from its vasodilatory effect, AM exerts diuretic5 and bronchodilatory effects,6 inhibits the release of aldosterone and adrenocorticotropic hormone,7 and contributes to the regulation of the proliferation, differentiation, and migration of a variety of cell types.8–10 Plasma AM levels are elevated during pregnancy,11 and placental tissues express high levels of AM,12 so that AM levels are also elevated in the amniotic fluid and in fetal membranes.13 Finally, plasma AM is elevated in the presence of such pathological conditions as hypertension, renal failure, heart failure, and shock.14–16
We recently generated a strain of AM knockout mice. Homozygotes (AM–/–) died in utero at approximately embryonic day (E) 13.5 to 14.0. The most apparent abnormality in AM–/– embryos at this stage was severe hemorrhage, which was observable under the skin and in the lung and liver. Hemorrhage was not yet detectable at E12.5 to E13.0, although histological examination showed the presence of poorly developed vitelline vessels on the yolk sac,17 which leads us to speculate that AM is indispensable for the development and/or maintenance of the vasculature during embryogenesis. In the present study, we further evaluate the angiogenic properties of AM using AM knockout mice, examining whether AM itself induces angiogenesis, or whether it promotes angiogenesis mediated by other factors.
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Materials and Methods |
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Unilateral Hind-Limb Ischemia Model
C3H mice (6-month-old, male) were randomly divided into 3 subgroups: an AM (Peptide Institute, Japan) group, a vascular endothelial growth factor (VEGF; Sigma) group, and a control group (Figure 1). All mice had experimentally induced hind-limb ischemia, produced by unilateral occlusion of the femoral arteries as described previously.18 The AM groups received a continuous subcutaneous injection of recombinant human AM dissolved in 0.9% saline at a rate of 50 ng/h using osmotic pumps (Alzet Model 2002) beginning the day before femoral occlusion. The VEGF group received an intramuscular injection of recombinant mouse VEGF (Sigma) dissolved in 0.9% saline containing 0.1% bovine serum albumin (5 ng/µL, 20 µL per mouse) into the ischemic limb soon after occlusion. The control group received a continuous injection of phosphate-buffered saline.
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, VEGF-treated group; 
, AM-treated group; 
, control group. D, Capillary density in the ischemic muscle 28 days after surgery. Data are expressed as fold increases relative to control, n=7 in each group. **P<0.01, *P<0.05 vs control group.Heterozygotic AM knockout mice (AM+/–) and their wild-type littermates (15- to 17-week-old, male 129/Sv-C57BL/6 hybrids) were also subjected to experimentally induced hind-limb ischemia. In addition, some of the wild-type mice were administered an AM antagonist, AM22-52 (Peptide Institute, Japan) (50 ng/h), using an osmotic pump, as was performed with AM (Figure 2).
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, AM+/– mice; All experiments were performed in accordance with the Declaration of Helsinki and were approved by the University of Tokyo Ethics Committee for Animal Experiments.
Blood Pressure Measurement
Blood pressures were measured using a programmable sphygmomanometer connected to a cuff probe for mice (MCP-1, Softron).19
Tumor Transplantation Model
Seventeen-week-old male ICR mice were divided into 5 groups: an AM group, a VEGF group, an AM22-52 group, a suramin group, and a control group (Figure 3A through 3E). Four of the groups were subcutaneously implanted with an osmotic pump filled with AM (50 ng/h), AM22-52 (100 ng/h), suramin (20 µg/h, Sigma), or phosphate-buffered saline (control), as per the respective group designation. Three days later, sarcoma 180 (S180) murine transplantable tumor cells were transplanted subcutaneously into the bilateral axillae of the mice at a dose of 2x106 cells in 0.2 mL per mouse. The remaining group of mice, which were not implanted with osmotic pumps, received an intratumor injection of recombinant mouse VEGF (5 ng/µL, 20 µL per tumor) 3 days after tumor transplantation. Heterozygotic AM knockout mice (AM+/–) and their wild-type littermates (15- to 17-week-old, male) were also subjected to transplantation with S180 cells (Figure 3F). In all cases, tumors were removed for analysis 10 days after transplantation.
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Laser Doppler Perfusion Imaging
Laser Doppler perfusion imaging (Moor Instruments Limited) was used to document hind limb and tumor blood flow, as reported previously.18,20 In the hind-limb ischemia models, laser Doppler perfusion imaging was performed soon after surgery and then on days 3, 7, and 12 thereafter. Calculated perfusion was expressed as a ratio of the left (ischemic) to the right (normal) limb.
Measurement of Capillary Density
Whole limbs were fixed in methanol overnight, after which the femora were removed and the ischemic thigh muscles were embedded in paraffin and sectioned. The sections were then deparaffinized and incubated with a rat monoclonal antibody against murine CD31 (Pharmingen). The distribution of antibody was visualized using the avidin–biotin complex technique and Vector Red chromogenic substrate (Vector Laboratories); sections were counterstained with hematoxylin. Capillaries were identified by positive staining for CD31 and by their morphology. Ten different fields from each muscle were randomly selected, and the number of capillaries was counted. Implanted tumors were removed and analyzed in the same way.
Analysis of VEGF Expression in the Ischemic Hind Limb
Western blot analysis of VEGF was performed using protein extracts from ischemic hind limbs obtained from control and AM-treated mice on days 0 (before femoral occlusion), 1 (24 hours after surgery), 3, 7, 14, 21, and 28. Extracts were also obtained from AM+/– mice and their wild-type littermates on day 12. The blots were probed with anti-VEGF antibody (0.5 µg/mL; Santa Cruz), as described previously,18 and with anti-ß-tubulin antibody (0.4 µg/mL; Santa Cruz), which served as an internal control.
In Vitro Model of Angiogenesis
The effect of AM (10–10 to 10–7 mol/L) administration on capillary formation in cocultures of human umbilical vein endothelial cells (HUVECs) and human diploid fibroblasts of dermal origin was evaluated21 using an angiogenesis assay kit (KZ-1000, Kurabo). VEGF (10 ng/mL) was added to the medium as a positive control. Capillary formation was confirmed microscopically, and the development and disposition of these structures were analyzed immunohistochemically to show expression of specific endothelial cell determinants (eg, CD31). Capillary length was measured by image analysis using an angiogenesis assay program (KSW-5000U, Kurabo).
Analysis of HUVEC and S180 Cell Proliferation
HUVECs were cultured for 24 hours in medium plus growth supplements and then for an additional 48 hours in medium with or without AM (10–7 mol/L), VEGF (10 ng/mL), or AM plus VEGF. S180 cells were cultured for 24 hours in medium with growth supplements and then in medium with or without AM (10–10 to 10–7 mol/L) for an additional 48 hours. The effect of AM on cell proliferation was then evaluated using cell counter (Coulter).
Analysis of VEGF Expression in Cultured Endothelial Cells
The effect of AM on VEGF expression in cultured human aortic endothelial cells was evaluated by reverse-transcription polymerase chain reaction (RT-PCR). Total RNA was prepared from human aortic endothelial cells using RNeasy mini kits (Qiagen) and reverse-transcribed. PCR was performed on the resultant cDNA samples using VEGF primers (GT) according to the manufacture’s recommendation. The primers for ß-actin were GCC GAT CCA CAC GGA GTA CT (sense) and CTG GCA CCC AGC ACA ATG (antisense).22 The amplification protocol entailed 2 minutes at 50°C and 10 minutes at 94°C, followed by 22 cycles of 15 seconds at 94°C and 1 minute at 60°C.
Analysis of Akt Activation in HUVECs
HUVECs were incubated for 1 hour in endothelial basal medium and then stimulated for 10 minutes with AM (0 to 10–6 mol/L), with or without VEGF (50 ng/mL), after which they were lysed in buffer. The lysates were subjected to SDS-PAGE, transferred to nitrocellulose membranes, and probed using anti-Akt and anti–phospho-Akt (p-Akt) antibodies (Cell Signaling Technology).
Restoration of Blood Flow by VEGF or AM in AM22-52–Treated and AM+/– Mice
To test whether blood flow recovery could be restored by administration of VEGF or AM in AM22-52–treated and AM+/– mice subjected to femoral artery occlusion, C3H mice (6-month-old, male) treated with AM22-52 (50 ng/h) were administered AM (50 ng/h) or VEGF (5 ng/µL, 20 µL) after surgery, after which blood flow recovery and capillary formation were compared with mice treated with AM22-52 alone and control mice (Figure 7A and 7B). In addition, some AM+/– mice (15- to 17-week-old, male 129/Sv-C57BL/6 hybrids) were also treated with AM or VEGF after surgery, and their blood flow recovery and capillary formation were compared with those in untreated AM+/– mice and their wild-type littermates (Figure 7C and 7D).
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Statistical Analysis
Values are expressed as means±SE. Student t tests and 1-factor ANOVA with post hoc tests were used to determine the significance of differences between means corresponding to each sample. Values of P<0.05 were considered significant.
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Results |
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Exogenous Administration of Recombinant AM Enhances Blood Flow Recovery
We used the hind-limb ischemia model to evaluate the angiogenicity of AM. Before unilateral femoral artery occlusion, systolic blood pressures in 6-month-old C3H mice averaged 102±1.8 mm Hg (n=7). On day 7, systolic blood pressures were lower in the AM-infused group, although the difference was not significant (108.4±2.0 mm Hg and 114.6±2.9 mm Hg, respectively; n=7 in each).
After femoral artery occlusion, autoamputation of ischemic toes was frequently observed in control mice (Figure 1A), whereas no autoamputation was observed in mice treated with AM or VEGF. This likely reflects the fact that the recovery of hind-limb blood flow was significantly better in VEGF- and AM-treated mice (Figure 1B and 1C). On day 12 after surgery, control mice showed 
70% blood flow recovery in the ischemic limb, whereas AM- and VEGF-treated mice showed almost full recovery (ratio of blood flow: control mice, 0.73±0.08; AM-treated mice, 0.98±0.05; VEGF-treated mice, 1.07±0.09; n=7 in each, P<0.05).
Formation of collateral vessels was then evaluated based on capillary density in samples of ischemic hind-limb muscle harvested 28 days after surgery. Anti-CD31 immunostaining confirmed that treatment with AM or VEGF significantly increased the number of capillaries in the ischemic limb (Figure 1D).
Capillary Formation Is Diminished in AM+/– Knockout Mice and AM Antagonist-Treated Mice
Heterozygous AM knockout (AM+/–) mice express 50% less AM than do wild-type mice.17 To investigate the role of endogenous AM in blood flow recovery, we also subjected AM+/– mice and their wild-type littermates to hind-limb ischemia. On day 7 after surgery, AM+/– mice showed 30% less blood flow recovery than did wild-type mice (blood flow ratios: wild-type, 1.0±0.10; AM+/–, 0.7±0.07; n=5 in each, P<0.05) (Figure 2A and 2B). Furthermore, on day 12, immunostaining for CD31 showed the capillary density in the ischemic limb of AM+/– mice to be significantly lower than in wild-type mice (Figure 2C).
To further confirm that a reduction in AM signaling actually impairs angiogenesis, we administered an AM antagonist, AM22-52, to the wild-type littermates of AM+/– mice. We found that blood flow recovery and capillary formation in the ischemic hind limb were significantly reduced by administration of AM22-52 (Figure 2B and 2C).
Tumor Angiogenesis Is Enhanced by AM and Reduced by AM22-52
In a set of parallel experiments, we also examined the role of AM in tumor angiogenesis. Ten days after transplantation of S180 tumor cells into mice, laser Doppler perfusion imaging showed that the blood flow at the transplantation site was increased in both AM-treated and control mice; however, the flow was much more pronounced in the former (Figure 3A). In addition, tumor weight (Figure 3B and 3C) and capillary density around and within the tumors were significantly greater in AM-treated animals (Figure 3D and 3E). Conversely, tumor weight and capillary density were significantly reduced in both AM22-52–treated (Figure 3C and 3E) and AM+/– (Figure 3F) mice.
We then cultured the tumor cells in vitro and confirmed that AM administration did not directly enhance tumor cell proliferation (cell proliferation ratios: control, 1.00±0.03; AM 10–9 M, 1.09±0.03; AM 10–8 M, 1.04±0.04; AM 10–7 M, 1.03±0.02; n=7 in each; NS), which supports the idea that the increased tumor growth seen in the AM-treated group was secondary to increased tumor angiogenesis.
Co-administration of AM and VEGF Enhances Capillary Formation In Vitro
Treatment with AM (10–7 mol/L) or VEGF (10 ng/mL) for 48 hours significantly enhanced proliferation of HUVECs. Moreover, co-administration of the same concentrations of AM and VEGF enhanced the proliferation more than either factor alone (Figure 4).
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Cocultures of HUVECs and fibroblasts formed capillary-like structures that were immunolabeled by anti-CD31 antibody (Figure 5A). Total capillary length calculated on day 11 after the start of the coculture revealed that capillary formation was significantly enhanced in the presence of VEGF (10 ng/mL) but not in the presence of AM alone (Figure 5B). However, co-administration of VEGF with AM (10–8 mol/L) enhanced capillary formation more than VEGF alone (Figure 5C).
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AM Administration Promotes VEGF Expression and Phosphorylation of Akt
With the aforementioned results in mind, we hypothesized that the increased angiogenesis seen in AM-treated mice might be related to the expression level of angiogenic factors. In the ischemic hind limb of AM-treated mice, the expression of VEGF was already upregulated on day 1 after femoral artery occlusion and remained elevated throughout the experimental period (Figure 6A). This suggests that with this model, AM primarily influences VEGF expression. Consistent with that idea, AM+/– mice showed diminished VEGF expression after 12 days of hind-limb ischemia (Figure 6B).
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Using cultured endothelial cells, we confirmed that AM administration dose- and time-dependently increased expression of VEGF (Figure 6C). In addition, when activation of the Akt pathway was analyzed in endothelial cells by evaluating levels of phospho-Akt (p-Akt), we found that although AM alone elicited minimal activation of the pathway, in the presence of VEGF (10 ng/mL), AM (10–11 to 10–7 mol/L) dose-dependently increased levels of p-Akt (Figure 6D).
VEGF or AM Administration Restores Blood Flow Recovery in AM22-52–Treated and AM+/– Mice
In the hind-limb ischemia model, AM22-52–treated and AM+/– mice showed less blood flow recovery and capillary formation than control or wild-type mice, respectively. We hypothesized that reduced VEGF signaling was the main cause of this impairment. To test that hypothesis, we determined the extent to which blood flow recovery could be restored by administration of VEGF or AM. We found that administration of VEGF or AM completely restored blood flow and capillary formation in both AM22-52–treated (Figure 7A and 7B) and AM+/– (Figure 7C and 7D) mice.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our findings indicate that AM exerts novel angiogenic effects that influence recovery of blood flow in ischemic tissue and tumor angiogenesis. Moreover, a role for endogenous AM in angiogenesis was implied by the findings that blood flow recovery in ischemic limbs and tumor angiogenesis are both impaired in AM+/– mice, which express only approximately half as much AM as do their wild-type littermates.
In the present study, we demonstrated that AM administration upregulates the expression of VEGF in both in vitro and in vivo models, which is consistent with the previously described angiogenic effect of AM noted by other groups using other models.23–24 We confirmed that AM dose- and time-dependently upregulates expression of VEGF in endothelial cells. Although AM alone failed to enhance capillary formation in a coculture system comprising endothelial cells and fibroblasts, capillary formation was enhanced by co-administration of AM and VEGF. In the presence of VEGF, AM dose-dependently promoted activation of the serine/threonine protein kinase Akt, which plays a central role in various models of angiogenesis and has been shown to mediate many of the biological effects of VEGF.25 AM may thus accelerate angiogenesis directly by inducing VEGF expression and by enhancing VEGF-mediated activation of the Akt-pathway. Consistent with this idea, the otherwise impaired angiogenesis in AM+/– and AM22-52–treated mice was restored by administration of VEGF or AM in the hind-limb ischemia model. Still, our experiment using a low-density, spotted array of angiogenic genes showed that AM administration also enhances the expression of other angiogenic factors, including endothelial nitric oxide synthase (data not shown), which is consistent with the earlier study of Abe et al,24 who suggested that NO is an important mediator of the angiogenic effects of AM.
AM was originally isolated from human pheochromocytoma as a vasodilating peptide;1 since then, it has been shown to be multifunctional, regulating the proliferation, differentiation, and migration of a variety of cell lines.8–10 In that regard, we recently showed that AM is indispensable for vascular morphogenesis during embryogenesis. Targeted null mutation of the AM gene is lethal in utero by E13.5 to 14.0, with the most apparent abnormality in surviving AM–/– mice being severe hemorrhage.17 We analyzed the arterial structure in AM–/– embryos and found that reduced expression of type IV collagen, which is produced by endothelial cells, is a major component of basement membrane and is implicated in the regulation of angiogenesis.26 AM is also reportedly involved in regulating the synthesis of the extracellular matrix.27 Its absence would therefore be expected to result in abnormal production and incorporation of extracellular matrix during formation of basement structures, which would in turn be expected to lead to the type of vessel malformation seen in AM–/– embryos.
Angiogenesis involves a complex series of events, during which endothelial cells locally degrade their basement membrane, migrate into the connective tissue stroma, proliferate at the migrating tip, elongate, and organize into capillary loops. Among the various mediators believed to be involved is VEGF,28 which is known to augment collateral blood flow in animals and patients with limb and myocardial ischemia.29 Although there was great hope for its clinical application, delivery of the VEGF gene alone causes fragile neovascularization, which leads to complications such as bleeding and microvascular leakage.30,31 Apparently, other mediators acting in concert with VEGF are necessary for physiologically intact vascular construction. Given its ability to induce expression of VEGF, enhance VEGF-induced Akt activity, and enhance production of a key basement membrane component, AM would seem to be a good candidate for such a mediator.
For our in vivo study, we chose the AM dosage and the treatment period of based on earlier studies. Nishikimi et al demonstrated that chronic infusion of AM using an osmotic pump effectively reduced glomerulosclerosis and improved renal function in Dahl salt-sensitive rats.32 They infused AM at a rate of 500 ng/h for 7 weeks, which raised plasma AM levels to within the physiological range (2 to 3 pmol/L). In addition, Yoshihara et al reported that chronic infusion of relatively small (130 g) rats with AM at a rate of 200 ng/h for 3 weeks attenuated monocrotaline-induced pulmonary vascular remodeling.33 In both cases, the AM dose/body weight ratio was approximately the same as that in the present study (1.5 ng/g body weight per hour), which we believed would be sufficient to modify cardiovascular remodeling.
High levels of AM expression have been detected in various types of cancer cells, suggesting that AM is involved in tumor growth;34–37 in fact, the presence of AM is associated with more aggressive tumor phenotypes in some cancer cell lines.38 This means that caution should be exercised when considering the possible therapeutic applications of AM, because one would not want to promote unfavorable angiogenesis associated with tumor growth. However, it means AM antagonists may effectively inhibit tumor growth through suppression tumor angiogenesis. We also found that AM22–52, a C-terminal fragment of AM and a selective AM receptor antagonist, suppressed tumor growth in vivo by suppressing angiogenesis.
In conclusion, we have shown that AM possesses novel angiogenic properties mediated by its ability to enhance VEGF expression and Akt activity. This may make it a useful therapeutic tool for relieving ischemia. Conversely, inhibitors of AM could be useful for clinical management of tumor growth.
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Acknowledgments |
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This work was supported by a grant-in-aid for Scientific Research from the Ministry of Education, Science, and Culture in Japan.
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Footnotes |
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Original received December 1, 2003; revision received June 21, 2004; accepted June 24, 2004.
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References |
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